Low Quantity single strand CAGE protocol

Hazuki Takahashi; Hiromi Nishiyori-Sueki; Piero Carninci

Jan 29, 2020

Low Quantity single strand CAGE protocol

DOI

dx.doi.org/10.17504/protocols.io.bbwkipcw

¹Laboratory for Transcriptome Technology, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa 230-0045, Japan

Hazuki Takahashi

Laboratory for Transcriptome Technology, RIKEN Center for In...

DOI: dx.doi.org/10.17504/protocols.io.bbwkipcw

Protocol Citation: Hazuki Takahashi, Hiromi Nishiyori-Sueki, Piero Carninci 2020. Low Quantity single strand CAGE protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bbwkipcw

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol in our group and it is working.

Created: January 29, 2020

Last Modified: January 29, 2020

Protocol Integer ID: 32428

Keywords: CAGE (Cap Analysis of Gene Expression), Transcription Starting Sites, Gene promoter, enhancer RNA,

Abstract

The Cap Analysis of Gene Expression (CAGE) is a powerful method to identify the Transcription Starting Sites (TSSs) of capped RNAs while simultaneously measuring transcript level. CAGE allows mapping at single nucleotide resolution all active promoters and enhancers. Large CAGE datasets have been over the years by individual labs and consortia, including the Encyclopedia of DNA Elements (ENCODE) and Functional Annotation Of the Mammalian Genome (FANTOM). These dataset constitute open resource for TSS annotations and gene expression analysis. 
Here we provide a protocol for the most recent CAGE version of the method, which is called Low Quantity (LQ) single strand (ss) CAGE (LQ-)ssCAGE. The methods allow to profile samples starting from low quantity of RNAs. Samples may typically include cells cultured in small volumes to maximizing large scale perturbations, cellular compartments like nuclear RNAs or samples from developmental stages. The advantage of (LQ-)ssCAGE is to allow working with small samples and simultaneously avoiding PCR amplification: this step help to preserve exquisite quantification of expression. The protocol makes use of simplified steps, by loading single stranded cap selected cDNA. Further, is allows reducing the RNA amount down to 25 ng and to shorten preparation time (less than 3 days) and to prepare libraries parallel, increasing the number of samples that one operator can reasonably handle (up to 96 samples). By proper organization of the flow of libraries preparation, an operator could further scale up the throughput. The obtained libraries are suitable for sequencing by various Illumina sequencing platforms using indexed reads and original barcode identifiers.

Materials

<Materials and Reagents>
Product name	                                                                                Maker	                                         Cat. No.
Agencourt AMPure XP	                                                                BECKMAN COULTER 	                 A63881
Agencourt RNAClean XP	                                                                BECKMAN COULTER 	                 A63987
KAPA Library Quantification Kits (500 reactions)-                         KAPA BIOSYSTEMS	                 KK4835 
Illumina / ABI Prism®	
Sodium periodate ACS Reagent Grade,	                                        MP Biomedicals	                      0215257725-25 g
5M Sodium Chloride, Molecular Biology Grade,	                        Promega	                                 V4221
RNase One Ribonuclease,	                                                                Promega	                                 M4261
Lithium chloride solution 8 M, for molecular biology, ≥99%	SIGMA-ALDRICH	                         L7026-100ML
Sodium acetate buffer solution,
BioXtra, pH 7.0±0.05 (25 °C), for molecular biology, 3 M,             SIGMA-ALDRICH	                         S2404-100ML
non-sterile; 0.2 µm filtered,	
Sodium periodate ACS Reagent Grade,	                                        SIGMA-ALDRICH	                         311448-5G
DNA Ligation Kit ＜Mighty Mix＞,                                                	TAKARA BIO INC.	                         6023
Ribonuclease H (RNase H) (20～60 U/µL),                                	TAKARA BIO INC.	                         2150A
10 mM dNTP Mix,                                                                        	ThermoFisher SCIENTIFIC          18427013
Dynabeads M-270 Streptavidin,                                                	ThermoFisher SCIENTIFIC	         65305
Ribonuclease H (2 U/µL),                                    	                        ThermoFisher SCIENTIFIC          18021014
RNaseZap RNase Decontamination Solution,	                        ThermoFisher SCIENTIFIC	         AM9780
SuperScript III Reverse Transcriptase,                                        	ThermoFisher SCIENTIFIC	         18080044
UltraPure 0.5 M EDTA, pH 8.0,                                                    	ThermoFisher SCIENTIFIC	         15575020
UltraPure DNase/RNase-Free Distilled Water,                                	ThermoFisher SCIENTIFIC	         10977015
Biotin (Long Arm) Hydrazide,                                                       	Vector Laboratories	                  SP-1100
0.5M EDTA (pH 8.0),                                                                    	Wako Pure Chemical Industries, 311-90075
10w/v% Polyoxyethylene(20) Sorbitan Monolaurate Solution,	Wako Pure Chemical Industries, 161-24801
1 M Tris-HCl (pH 7.0),                                                                    	Wako Pure Chemical Industries, 311-90411　
1M Tris-HCl (pH 7.5),                                                                    	Wako Pure Chemical Industries, 316-90221
1M Tris-HCl (pH 8.5),                                                                    	Wako Pure Chemical Industries, 316-90405
3M Sodium Acetate (pH 5.2),                                                    	Wako Pure Chemical Industries, 316-90081
Dimethyl Sulfoxide,                                                                        	Wako Pure Chemical Industries, 046-21981
Ethanol (99.5),                                                                         	        Wako Pure Chemical Industries, 057-00456
2M NaOH,                                                                        	                Wako Pure Chemical Industries, 194-05631　
Hybridization buffer HT1,                                                         　	Illumina

<Equipment>　	　　
Product name,	                                                                                Maker	                                            Cat. No.
Axygen 0.2 mL Polypropylene PCR Tube Strips                             CORNING, AXYGEN	                PCR-0208-CP-C
and Domed Cap Strips, 8 Tubes/Strip, 8 Domed Caps/Strip, 
Clear, Nonsterile	
Axygen 1.5 mL Maxymum Recovery Snaplock                               CORNING, AXYGEN	                 MCT-150-L-C
Microcentrifuge Tube, Polypropylene, Clear, Nonsterile, 
250 Tubes/Pack, 10 Packs/Case	
Axygen 16 Well Polypropylene PCR Microplate,                             CORNING, AXYGEN	                 PCR-16-C
Clear, Nonsterile	
Axygen PCR 1 x 8 Strip Domed Caps,                                               CORNING, AXYGEN	                  PCR-02CP-C
Fit 0.2 mL PCR Tube Strips, Clear, Nonsterile,	
X-Pierce Sealing Films, Sterile,	                                                         EXCEL Scientific, Inc.	                  XPS-25
10 µL (0.1 - 10 µL) LongReach Barrier tip,	                                 Sorenson BioScience, Inc.	          38000T
10 µL (0.1 - 10 µL) Ultra G Barrier tip,	                                         Sorenson BioScience, Inc.	          28200T
1000 µL (100-1000 µl) Barrier tip,	                                                 Sorenson BioScience, Inc.	          14200T
20 µL (1-20 µL) Barrier tip,	                                                                 Sorenson BioScience, Inc.	          14210T
200 µL (1-200 µL) NX Barrier tip,	                                                 Sorenson BioScience, Inc.	          30550T
MicroAmp Optical 384-Well Reaction Plate with Barcode,	         ThermoFisher SCIENTIFIC	  4309849
MicroAmp Optical Adhesive Film,	                                                 ThermoFisher SCIENTIFIC	  4360954
PIPETMAN Classic, P1000,                                                                 GILSON	                                          F123602
PIPETMAN Classic, P2,	                                                                 GILSON	                                          F144801
PIPETMAN Classic, P20,          	                                                         GILSON	                                          F123600
PIPETMAN Classic, P200,                                	                                 GILSON	                                          F123601
MJ Research Tetrad PTC-225 Thermal Cycler,                        	 GMI, MJ Research	                  8252-30-1004
MINI CENTRIFUGE,　	                                                                         HARMONY (LMS)	                  CFM-1000
PlateSpin II,                                                                                     	 KUBOTA	　
Proline Plus Mechanical Pipettes, 8-ch, 0.5 - 10 µL,            	         sartorius	                                   728120
Proline Plus Mechanical Pipettes, 8-ch, 10 - 100 µL,	                 sartorius	                                   728130
Proline Plus Mechanical Pipettes, 8-ch, 30 - 300 µL,	                 sartorius	                                   728140
Vortex-Genie 2,	                                                                                 Scientific Industries, Inc.	           SI-0286
miVAC DNA,	                                                                                         SP Scientific, Genevac	           DNA-10000-G00
miVac rotor for micro plate,	                                                         SP Scientific, Genevac	           DRS-00000-200
Dynabeads MPC-S (Magnetic Particle Concentrator),    	        ThermoFisher SCIENTIFIC	           A13346
DynaMag-96 Side Skirted Magnet,	                                                ThermoFisher SCIENTIFIC	           12027


<RT primer (for ssCAGE)>
name	    5’ modification	    Sequence	                3’ modification 	base
N6+TCT    	P	                    TCTNNNNNN		                                  9


<RT primers containing barcode (for LQ-ssCAGE)>
name	                      5’ modification	Sequence	                                                                          	  Base                                                                               
TCT_7nt_N15_#001	P	                        TCTGATGCTCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#002	P	                        TCTTATGAGCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#003	P	                        TCTCGACGATNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#004	P	                        TCTTAGTCACNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#005	P	                        TCTCGTACTGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#006	P	                        TCTTACGTAGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#007	P	                        TCTAGACTCGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#008	P	                        TCTTCGCTGANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#009	P	                        TCTCGATCTGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#010	P	                        TCTTGTCACGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#011	P	                        TCTATGCACTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#012	P	                        TCTGACATCGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#013	P	                        TCTATCTAGCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#014	P	                        TCTTGTCGCANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#015	P	                        TCTCAGATCTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#016	P	                        TCTTCAGAGCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#017	P	                        TCTATACTGCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#018	P	                        TCTATCGTGANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#019	P	                        TCTGAGCTCTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#020	P	                        TCTTCTCGAGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#021	P	                        TCTCAGTGTCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#022	P	                        TCTGCTACGANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#023	P	                        TCTAGTACGCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#024	P	                        TCTGACTAGCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#025	P	                        TCTTACGCTANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#026	P	                        TCTGCTATAGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#027	P	                        TCTACACTGTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#028	P	                        TCTACATGTCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#029	P	                        TCTGCAGACTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#030	P	                        TCTTAGCGACNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#031	P	                        TCTACAGTCGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#032	P	                        TCTGTATGACNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#033	P	                        TCTCAGCAGTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#034	P	                        TCTACAGCTGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#035	P	                        TCTGCTGATANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#036	P	                        TCTTGTCGACNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#037	P	                        TCTTGCGATANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#038	P	                        TCTAGTGTCANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#039	P	                        TCTATACGTCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#040	P	                        TCTCACTATGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#041	P	                        TCTTCACAGTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#042	P	                        TCTTACGCGTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#043	P	                        TCTCGACGTANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#044	P	                        TCTAGCATGCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#045	P	                        TCTATCGCGTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#046	P	                        TCTGATATGCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#047	P	                        TCTTAGCATCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#048	P	                        TCTGCTCATGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#049	P	                        TCTCTGATGCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#050	P	                        TCTGCATATGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#051	P	                        TCTGTCACATNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#052	P	                        TCTCTAGCATNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#053	P	                        TCTCACGTGTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#054	P	                        TCTCTAGTACNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#055	P	                        TCTGATCGTANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#056	P	                        TCTCTAGTCGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#057	P	                        TCTGACACTGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#058	P	                        TCTAGCTCGANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#059	P	                        TCTGACTGCANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#060	P	                        TCTAGACGCTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#061	P	                        TCTCAGTCTGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#062	P	                        TCTTGACTGCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#063	P	                        TCTATCAGCGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#064	P	                        TCTCTCAGAGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#065	P	                        TCTTATGCACNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#066	P	                        TCTTGCTACANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#067	P	                        TCTATGCGCANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#068	P	                        TCTTCGTGACNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#069	P	                        TCTCGATACGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#070	P	                        TCTGCTAGCANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#071	P	                        TCTTAGCGTANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#072	P	                        TCTCTGACGANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#073	P	                        TCTTACGTCANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#074	P	                        TCTCATAGCTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#075	P	                        TCTTCGATCANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#076	P	                        TCTCATATCGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#077	P	                        TCTCTCGATANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#078	P	                        TCTAGTCAGTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#079	P	                        TCTTCGATAGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#080	P	                        TCTGCGTATANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#081	P	                        TCTATGTCAGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#082	P	                        TCTACGTCGANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#083	P	                        TCTCACTAGTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#084	P	                        TCTTCACGTGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#085	P	                        TCTGCTGTACNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#086	P	                        TCTTACTGAGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#087	P	                        TCTCGATGCANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#088	P	                        TCTTGTCAGANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#089	P	                        TCTCTCTAGANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#090	P	                        TCTCGTACATNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#091	P	                        TCTCTAGCTGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#092	P	                        TCTCGCTATANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#093	P	                        TCTTAGCTCANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#094	P	                        TCTGTATAGCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#095	P	                        TCTGTGACTCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#096	P	                        TCTAGCATCGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#097	P	                        TCTTGAGCATNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#098	P	                        TCTCGTATAGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#099	P	                        TCTACTAGCGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#100	P	                        TCTACTGATGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#101	P	                        TCTGTACATGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#102	P	                        TCTTGACTCANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#103	P	                        TCTGATCTCANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#104	P	                        TCTCGACTGTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#105	P	                        TCTCAGTAGCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#106	P	                        TCTACTATGCNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#107	P	                        TCTACGATCGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#108	P	                        TCTGATCACGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#109	P	                        TCTTGCATAGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#110	P	                        TCTCGTCATANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#111	P	                        TCTAGCTCAGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#112	P	                        TCTGTAGCACNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#113	P	                        TCTCTATGCGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#114	P	                        TCTGACTACTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#115	P	                        TCTGATCGCTNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#116	P	                        TCTCTGATAGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#117	P	                        TCTGTGCATANNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#118	P	                        TCTGATGCATNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#119	P	                        TCTACTGCAGNNNNNNNNNNNNNNN		                            25
TCT_7nt_N15_#120	P	                        TCTTGATCACNNNNNNNNNNNNNNN		                            25


<5’ linkers> 
name, 5’ modification, sequence, 3’ modification, base
5’adaptor N6, -, AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNN, P,	64
5’adaptor GN5, -, AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGNNNNN, P, 64
5’adaptor down, P, AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT, P, 58

<3’ linkers >
name		
sequence, 3’ modification,	base
3’end adaptor up 01		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG, P, 67
3’end adaptor down 01		CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 61
3’end adaptor up 02		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG, P, 67
3’end adaptor down 02		CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 61
3’end adaptor up 03		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCATCTCGTATGCCGTCTTCTGCTTG, P, 67
3’end adaptor down 03		CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 61
3’end adaptor up 04		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTG, P, 67
3’end adaptor down 04		CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 61
3’end adaptor up 05		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTTCTGCTTG, P, 67
3’end adaptor down 05		CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 61
3’end adaptor up 06		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCAATATCTCGTATGCCGTCTTCTGCTTG, P, 67
3’end adaptor down 06		CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 61
3’end adaptor up 07		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCTGCTTG, P, 67
3’end adaptor down 07		CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 61
3’end adaptor up 08		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACACTTGAATCTCGTATGCCGTCTTCTGCTTG, P, 67
3’end adaptor down 08		CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 61
3’end adaptor up 09		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACGATCAGATCTCGTATGCCGTCTTCTGCTTG, P, 67
3’end adaptor down 09		CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 61
3’end adaptor up 10		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACTAGCTTATCTCGTATGCCGTCTTCTGCTTG, P, 67
3’end adaptor down 10		CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 61
3’end adaptor up 11		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCTACATCTCGTATGCCGTCTTCTGCTTG, P, 67
3’end adaptor down 11		CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 61
3’end adaptor up 12		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGTAATCTCGTATGCCGTCTTCTGCTTG, P, 67
3’end adaptor down 12		CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 61
3’end adaptor up 13		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTCAACAATCTCGTATGCCGTCTTCTGCTTG, P, 69
3’end adaptor down 13		CAAGCAGAAGACGGCATACGAGATTGTTGACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 63
3’end adaptor up 14		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTTCCGTATCTCGTATGCCGTCTTCTGCTTG, P, 69
3’end adaptor down 14		CAAGCAGAAGACGGCATACGAGATACGGAACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 63
3’end adaptor up 15		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACATGTCAGAATCTCGTATGCCGTCTTCTGCTTG, P, 69
3’end adaptor down 15		CAAGCAGAAGACGGCATACGAGATTCTGACATGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 63
3’end adaptor up 16		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGTCCCGATCTCGTATGCCGTCTTCTGCTTG, P, 69
3’end adaptor down 16		CAAGCAGAAGACGGCATACGAGATCGGGACGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 63
3’end adaptor up 18		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACGTCCGCACATCTCGTATGCCGTCTTCTGCTTG, P, 69
3’end adaptor down 18		CAAGCAGAAGACGGCATACGAGATGTGCGGACGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 63
3’end adaptor up 19		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGAAACGATCTCGTATGCCGTCTTCTGCTTG, P, 69
3’end adaptor down 19		CAAGCAGAAGACGGCATACGAGATCGTTTCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 63
3’end adaptor up 20		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGGCCTTATCTCGTATGCCGTCTTCTGCTTG, P, 69
3’end adaptor down 20		CAAGCAGAAGACGGCATACGAGATAAGGCCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 63
3’end adaptor up 21		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTTCGGAATCTCGTATGCCGTCTTCTGCTTG, P, 69
3’end adaptor down 21		CAAGCAGAAGACGGCATACGAGATTCCGAAACGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 63
3’end adaptor up 22		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCGTACGTAATCTCGTATGCCGTCTTCTGCTTG, P, 69
3’end adaptor down 22		CAAGCAGAAGACGGCATACGAGATTACGTACGGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 63
3’end adaptor up 23		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACGAGTGGATATCTCGTATGCCGTCTTCTGCTTG, P, 69
3’end adaptor down 23		CAAGCAGAAGACGGCATACGAGATATCCACTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 63
3’end adaptor up 25		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACACTGATATATCTCGTATGCCGTCTTCTGCTTG, P, 69
3’end adaptor down 25		CAAGCAGAAGACGGCATACGAGATATATCAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 63
3’end adaptor up 27		NNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACATTCCTTTATCTCGTATGCCGTCTTCTGCTTG, P, 69
3’end adaptor down 27		CAAGCAGAAGACGGCATACGAGATAAAGGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGA, - , 63

<Recipes>
1.  250 mM NaIO4
Dissolve 1 mg of Sodium periodate (ACS Reagent Grade) in 18.7 µL of RNase/DNase free water and keep in the dark. NOTE: The solution can be aliquoted to 50 µL and stored at 80 °C.

2.  100 mM Biotin (long arm) Hydrazide
Dissolve 50 mg of Biotin (long arm) Hydrazide in 1.345 mL of DMSO 1.345ml.
Biotin is packed in 50mg/tube. 
NOTE: The solution can be aliquoted to 50 µL and stored at 80 °C.

3. LiCl buffer
Mix following materials and store at room temperature.

8M Lithium chloride solution					                          3.64 mL
1 M Tris-HCl (pH 7.5)					                                          0.80 mL
10w/v% Polyoxyethylene (20) Sorbitan Monolaurate Solution	  0.40 mL
0.5 M EDTA (pH 8.0)					                                          0.16 mL
RNase, DNase –free water					                                  3.64 mL

4. TE wash buffer
Mix following materials and store at room temperature.

RNase, DNase –free water					                                  9.12 mL
1 M Tris-HCl (pH 7.5)					                                          0.40 mL
10w/v% Polyoxyethylene (20) Sorbitan Monolaurate Solution	  0.40 mL
0.5 M EDTA (pH 8.0)					                                          0.08 mL

5. Release buffer
Mix following materials and store at room temperature.

RNase ONE 10x Reaction Buffer				                        100 µL
10w/v% Polyoxyethylene (20) Sorbitan Monolaurate Solution	 1.0 µL
RNase, DNase –free water					                                899 µL

6. 1 x TE buffer
Mix following materials and store at room temperature.

1 M Tris-HCl (pH 8.0)				500 µL
0.5 M EDTA (pH 8.0)				100 µL
RNase, DNase-free water 			49.4 mL

7. 0.1 M NaCl/TE buffer
1 M NaCl 						500 µL
1 x TE buffer 					4.5 mL 

8. 2.5 µM 5’linker 
a. Mix following solution and carry out the annealing reaction to generate 100 µM GN5 linker

1 mM 5’adaptor GN5	 4 µL　　　
1 mM 5’adaptor down	 4 µL
1 M NaCl		         4 µL
1 x TE buffer		       28 µL

<Annealing condition>
95 °C, 5 min: − 0.1 °C sec −1 down to 83 °C; 5 min at 83 °C; − 0.1 °C sec−1 down to 71 °C; 5 min at 71 °C; − 0.1 °C sec−1 down to 59 °C; 5 min at 59 °C; −0.1 °C sec−1, to 47 °C; 5 min at 47 °C; −0.1 °C sec−1, to 35 °C; 5 min at 35 °C; −0.1 °C s−1 to 23 °C; 5 min at 23 °C; −0.1 °C sec−1 to 11 °C, and then hold at 11 °C.

b. Mix following solutions and repeat the annealing reaction at step a. to generate 100 µM N6 linker

1 mM 5’adaptor N6	1 µL
1 mM 5’adaptor down	1 µL
1M NaCl		                1 µL
1 x TE buffer		        7 µL

c. Mix 40 µL of 100 µM GN5 linker from step a. and 10 µL of 100 µM N6 linker from step b.
d. Dilute 100 µM of mixed 5’linkers to 2.5 µM in 0.1 M NaCl/TE buffer. For instance, add 2.5 µL of 100 µM mixed linkers from step c to 97.5 µL of 0.1 M NaCl/TE buffer for 25 samples.

NOTE: 2.5 µM and 100 M mixed 5’linkers can be stored at -20°C.

9. 2.5 µM 3’ linker
a. Mix following solutions and carry out the annealing reaction at the step 8.a. to generate 100 µM of 3’ linker.

1 mM 3’end adaptor up	        1 µL
1 mM 3’end adaptor down	1 µL
1 M NaCl		        1 µL
1 x TE buffer		        7 µL

b. Dilute 100 µM mixed linker from step a. to 2.5 µM in 0.1 M NaCl/TE buffer (see details at the step 8.d.).

Before start

RNAs should be treated in the RNAse free solutions, benches and equipments. We highly recommend to perform RNA quality check before starting (see detail at previously published paper RNA stability check).

Figure.1 Protocol Flow

Reverse Transcription (RT) to generate RNA/cDNA hybrids
 

single strand CAGE (ssCAGE)
When the amount of RNAs is more than1 µg, choose this RT protocol. Here is an example of 5 µg starting RNAs.
When the amount of RNAs is less than 1 µg, go to step 1.2.

a. Mix 5 µL of RNAs (1 µg/µL), 1 µL of RT primer (N6+TCT) and 4 µL of RNase free water in a tube.
Note
NOTE: Use 1.5 mL tube, 8 strip PCR tube or 16-96 wells plate  depending on numbers of samples.

b. Incubate the RNA/primer mix from step a. at 65 °C for 5 min and immediately put on ice for 2 min.
c. Add the following components to the solution from step b. and carefully mix by pipetting on ice. 

5 x First-Strand buffer                             4 µL
10 mM dNTPs                                          1 µL
0.1 M DTT                                                 1 µL
RNase free water                                     2 µL
SuperScript III Reverse Transcriptase  2 µL

d. Incubate the mixed solution (total 20 µL) at 25 °C for 30 sec, 55 °C for 30 min and keep 4 °C to generate RNA/cDNA hybrids.
e. Add 36 µL (1.8-folds) of RNACleanXP beads to 20 µL of RNA/cDNA hybrids from step d. and mix well by pipetting. 
①. Incubate at room temperature for 5 min. Spin down and set the tube on magnetic bar for 5 min. 
②. Discard the supernatant by pipette aspiration. 
③. Wash the beads with 200 µL of 70% EtOH. 
④. Set the tube on magnetic bar for 5 min. 
⑤. Discard the supernatant by pipette aspiration. 
⑥. Repeat step ③ ~ ⑤.
⑦. Discard the 70% EtOH completely by pipette aspiration. 
⑧. Add 42 µL of RNase free water and mix by pipetting extensively (more than 60 times) to elute RNA/cDNA hybrids.
⑨. Incubate at room temperature for 5 min. 
⑩. Spin down and set the tube on magnetic bar for 5min. 
⑪. Transfer supernatant (~ 40 µL of the cDNA/RNA hybrids) to new tube. 

Low Quantity single strand CAGE (LQ-ssCAGE)
When the amount of starting RNAs is less than 1 µg, choose this protocol. 
Note

NOTE: The amount of RNAs in one tube should be 25 ng ~ 100 ng for the RT reaction. After RT, samples have each barcode and can be mixed up to 5 µg. For instance, if the starting RNA amount is 50 ng, 100 samples can be mixed. The number of mixed samples is depending on how much samples you have and how deep the samples should be sequenced. Here is an example of 48 mix samples from each 50 ng starting RNA.
CRITICAL: The mixed solution should be less than 5 µg. Otherwise the following linkers at step 9 and 11 are not enough for the linker ligation. 


a. Mix 4 µL of 50 ng RNAs (12.5 ng/µL) and 1 µL of 1.25 mM RT primer containing barcode in a 96 wells plate by pipetting. 
b. Incubate the RNA/primer mix form step a. at 65 °C for 5 min and immediately put on ice for 2 min.
c. Mix the following components (enzyme mix) on ice: 

5 x First Strand buffer                             2.0 µL 
0.1 M DTT                                                 0.5 µL
10 mM dNTPs                                          0.5 µL
RNase free water                                     1.0 µL
SuperScript III Reverse Transcriptase  1.0 µL
Total volume                                             5.0 µL

Note
NOTE: Make premix solution for 48 samples x 1.1 samples.


d. Add 5 µL of enzyme mix from premix solution at step c. to RNA/primer mix from step b. and carefully mix 10 times by pipetting on ice.
e. Incubate at 25 °C for 30 sec, 50 °C for 30 min and keep 4 °C to generate RNA/cDNA hybrids.
f. Mix samples by following steps. 
①. Transfer each 10 µL of RNA/cDNA hybrids from step e. to one of new 1.5 mL tube (total volume is 480 µL). 
②. Add 15 µL of RNase free water to the first 8 wells at the 96 wells plate from step ①, wash wells by pipetting, and transfer the 15 µL of the solutions to the next 8 wells. 
③. Wash the 8 wells by pipetting and transfer 15 µL of the solutions to the next 8 wells. 
④. Repeat step ②three times until the end of 8 wells and transfer all solutions (total volume is 120 µL (15 µL x 8 wells)) to the 1.5 mL tube from step ①(final volume is 600 µL).
⑤. Mix 600 µL of the solution from step ④by vortex, spin down and aliquot 200 µL in new two tubes of 1.5 mL tube (total 200 µL in 3 tubes of 1.5 mL tube). 
Note
NOTE: Step f. is for the collection of remaining molecules to avoid losing any RNA/cDNA hybrids in the wells. We recommend to aliquot less than 200 µL per each 1.5 mL tube at step f.⑤due to the volume limitations of next RNAClean XP purification step. 

g. Add 300 µL (1.8 -folds) of RNAClean XP beads to the 48 mixed RNA/cDNA hybrids in a 1.5 mL tube from step f.⑤, mix well by pipetting and then elute the mixed RNA/cDNA hybrids by following steps.
①. Incubate at room temperature for 10 min, spin down and set the 1.5 mL tube on magnetic bar for 5 min. 
②. Discard the supernatant by pipette aspiration. 
③. Wash the beads with 1.2 mL of 70% EtOH. 
④. Set the 1.5 mL tube on magnetic bar for 5 min. 
⑤. Discard the supernatant by pipette aspiration. 
⑥. Repeat step ③~ ⑤ twice.
⑦. Discard the 70% EtOH completely by pipette aspiration. 
⑧. Add 100 µL RNase free water and mix by pipetting extensively (more than 60 times) to elute RNA/cDNA hybrids.
⑨. Incubate at room temperature for 5 min. 
⑩. Spin down and set the tube on magnetic bar for 5min. 
⑪. Transfer 100 µL of the cDNA/RNA hybrids to new 1.5 mL tube. 
⑫. Repeat step ⑧ ~ ⑪ twice (final volume is 200 µL). 
⑬. Concentrate 200 µL of the cDNA/RNA hybrids solutions from step ⑫ to around 40 µL by SpeedVac vacuum concentrator at 37 °C, and collect the solutions from 3 of 1.5 mL tubes to 1 of 1.5 mL tube (total volume is around 120 µL) and concentrate to 40 µL in the 1.5 mL tube at 37°C. The timing is around 2 h. 
Note
CRITICAL: DO NOT DRY UP the cDNA/RNA hybrids solutions by the concentrating step ⑬. Check the sample volume during the concentration several times and adjust the final volume to 40 µL with RNase/DNase-free when the volume become less than 40 µL.

Oxidation to modify diol group of cap structure

a. Mix 40 µL of RNA/cDNA hybrid from step 1.1e. or 1.2g., 2 µL of 1 M NaOAc (pH 4.5) and 2 µL of 250 mM NaIO4 in a tube by 10 times pipetting.
Note
NOTE: 1.5 mL tube, 8-Strip PCR tube or16 wells plate can be used depending on how many samples should be prepared. 

b. Incubate for 5 min on ice in dark by aluminum foil wrapping.
c. Add 16 µL of 1 M Tris-HCl (pH 8.5) to neutralize the solution and mix well by pipetting.

Purification

Add 108 µL (1.8 -folds) of RNACleanXP beads to 60 µL of oxidated RNA/cDNA hybrid from step 2, mix well by pipetting and then elute the 48 samples mixed RNA/cDNA hybrids by following steps.
①. Incubate at room temperature for 5min.
②. Spin down and set the tube on magnetic bar for 5 min. 
③. Discard the supernatant by pipette aspiration. 
④. Wash the beads with 200 µL of 70% EtOH 
⑤. Discard the 70% EtOH. 
⑥. Repeat step ④~ ⑤twice and discarded 70% EtOH completely.
⑦. Add 42 µL of RNase free water and mix by pipetting extensively (more than 60 times) to elute supernatant. 
⑧. Incubate at room temperature for 5min. 
⑨. Spin down and set the tube on magnetic bar for 5 min. 
⑩. Collect 40 µL of the supernatant to new tube.

Biotinylation by the coupling reaction to the oxidized RNA/cDNA hybrids

a. Mix 40 µL of purified oxidized RNA/cDNA hybrid from step 3, 4 µL of 1M NaOAc (pH 6.3) and 4 µL of 100 mM Biotin (long arm) hydrazide by 10 times pipetting.
b. Incubate for 30 min at 40 °C.
c. Add 86.4 µL of RNACleanXP (1.8 -folds) to 44 µL of solution from step b, and perform purification by following step 3 ① ~ ⑩. 
Note
NOTE: Biotinylation happens both 5’end of capped RNAs and 3’end of RNA (see Figure 2 at published protocol).

RNaseONE treatment to digest RNA of RNA/cDNA hybrids

a. Add 4.5 µL of 10 x RNaseONE buffer and 0.5 µL of RNaseONE to 40 µL of purified biotinylated RNA/cDNA hybrids from step 4 and mix 10 times by pipetting. 
b. Incubate for 30 min at 37 °C.
c. Add 81 µL of RNACleanXP (1.8 -folds) to the solution from step b. and purify by the following step 3 ①~ ⑩.

Note
CRITICAL: This step is critical to digest uncompleted cDNA synthesis to the 5’ end of capped RNAs (See Figure 2 at published protocol).

Dynabeads M-270 Streptavidin beads preparation

a. Add 30 µL of Dynabeads M-270 Streptavidin to new 1.5ml tube, set on the magnetic bar for 5 min, and discard supernatant. 
b. Wash the beads with 30 µL of LiCl buffer and discard supernatant.
c. Repeat step b. for 2 times.
d. Resuspend the beads in 35 µL of LiCl buffer.

CapTrap reaction

a. Add 35 µL of beads from step 6 to 40 µL of RNA/cDNA hybrids from step 5 and mix well by pipetting.
b. Incubate for 15 minutes at 37 °C and mix by pipetting at the time of 7 min. 
c. Spin down and set the plate on the magnetic bar for 2 min and discard the supernatant by the pipette aspiration.
d. Add 150 µL of TE wash buffer and mix 60 times by pipetting. Spin down and set the plate on the magnetic bar for 2 min and discard the supernatant. 
e. Repeat step d. for 3 times.
f. Add 35 µL of Release buffer to the beads and mix 60 times by pipetting. 
g. Incubate for 5 min at 95°C for 5min and on ice for 1 min. 
h. Spin down and set the tube on the magnetic bar for 2 min.
i. Transfer the supernatant containing CapTrapped cDNA from step h. to new 16 well plate. 
j. Add 30 µL of Release buffer to the beads and mix well by pipetting, spin down, and set the tube on the magnetic stand for 2 min. 
k. Transfer 30 µL of supernatant containing CapTrapped cDNA to new tube.

RNaseONE and RNaseH reaction to remaining RNAs
a. Add 2.9 µL of release buffer, 2 µL of RNaseONE and 0.1 µL of RNase H to 30 µL of CapTrapped cDNA from step 7 and mix 10 times by pipetting.
b. Incubate at 37°C for 30 min.
c. Add 126 µL of AMPureXP (1.8 -folds) to 33 µL of cDNA from step b. and perform purification by following step 3 ① ~ ⑩.
d. Dry up 40 µL of purified CapTrapped cDNA by the SpeedVac concentrator at 37°C for around 75 min. 
e. Add 4 µL of RNase free water to the dried pellet.

5' Single Strand Linker Ligation

a. Incubate 4 µL of cDNA at 95 °C for 5 min and put on ice for 2 min. 
b. Incubate 4 µL of 5’ linker at 55 °C for 5 min and put on ice for 2 min. 
c. Mix 4 µL of 5’ linker, 4 µL of CapTrapped cDNA from step a. and 16 µL of Mighty Mix by pipetting (total volume is 24 µL). 
d. Incubate at 16 °C for 16 h (overnight).

Purification to remove excess 5’ linkers and linker dimers

a. Add 46 µL of RNase free water and 126 µL of AMPureXP (1.8 -folds) to 24 µL of cDNA from step 9, mix well by pipetting.
b. Purify the solution by following step 3 ①~ ⑩.
c. Incubate at 95 °C for 5 min and immediately put on ice for 2 min.
d. Add 48 µLof AMPureXP beads (1.2 -folds) to 40 µL of cDNA and mix well by pipetting. 
e. Repeat purification by following step 3 ① ~ ⑩.
f. Dry up 40 µL of cDNA by SpeedVac concentrator at 80°C for around 35 min. 
g. Add 4 µL of RNase free water to the dried pellet.

Note
CRITICAL: It is important to perform heat inactivation and repeat purification step to remove non-ligated excess linkers during the purification step. Otherwise, these excess linkers can disturb library sequencing quality; excess linkers compete with the library to anneal Illumina flow-cells. In addition, 5’linker-3’linker dimers can be created at the next step 11. 

3’ Single Strand Linker Ligation
a. Incubate 4 µL of cDNA at 95 °C for 5 min and put on ice for 2 min. 
b. Incubate 4 µL of 3’ linker at 65 °C for 5 min and put on ice for 2 min. 
c. Mix 4 µL of 3’linker, 4 µL of cDNA from step a. and 16 µL of Mighty Mix by pipetting. 
d. Incubate at 30 °C for 4 h.
Note
NOTE: When you have more than 2 libraries with same barcode at RT primer and plan to mix libraries at the sequencing step 14, use different Index at the 3'linker. 

Purification to remove excess 3’ linkers and linker dimers (final library)
a. Purify the solution from step 11 by following step 11a.-f.
b. Dissolve the pellet with 10 µL of RNase, DNase-free water.

 

Library QC
Quantify the concentration of library by KAPA Library Quantification Kit.

Sequencing
a. Mix the following components 

Library (2250 amol)                    X µL
RNase, DNase-free water  (19-X) µL
2N NaOH                                       1 µL
Total                                             20 µL

b. Incubate at room temperature for 5 min.
c. Put the tube on ice and add 20 µL of 1 M Tris-HCl (pH 7.0, pre-chilled) to neutralize.
d. Add 110 µL of HT1 buffer.
e. Transfer 120 µL of the denatured and diluted library to HiSeq2500.

Sequencing kit  
ssCAGE: 50 bp Single Read sequencing.
LQ-ssCAGE: 50 bp Paired-End sequencing (see Figure 1 for the library structure).
       Read 1: to read sequence information of cDNA 
       Read 2: to read sequence information of barcode in RT primer
       Index 1: to read sequence information of index at the 3'linker    

Public workspaceLow Quantity single strand CAGE protocol

Low Quantity single strand CAGE protocol