Apr 18, 2025
JAX_DPC: Library preparation using KAPA RNA HyperPrep Kit for Bulk RNAseq
- Christine Goldfarb1,
- Jennifer Idol1,
- Rahul Maurya1,
- chewyee.ngan1,
- Mark Adams1
- 1The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA
- MorPhiC Consortium
Protocol Citation: Christine Goldfarb, Jennifer Idol, Rahul Maurya, chewyee.ngan, Mark Adams 2025. JAX_DPC: Library preparation using KAPA RNA HyperPrep Kit for Bulk RNAseq. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4z39ovo5/v1
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 and it's working
Created: April 17, 2025
Last Modified: April 18, 2025
Protocol Integer ID: 130941
Funders Acknowledgements:
JAX MorPhiC DPC
Grant ID: 1 UM1 HG012651-01
Abstract
This protocol describes the preparation and sequence of libraries for bulk RNAseq with RNA extracted from cortical brain organoids or extra-embryonic derived iPSCs. Herein we use KAPA RNA HyperPrep Kit with RiboErase (HMR) from Roche for library construction and NovaSeq X Plus from Illumina.
Procedure
Procedure
RNA extraction from cell pellet
For regular samples use the RNase-Free DNase Set from Qiagen to extract RNA according to the manufacturer’s protocol:
For samples with lower cell count use the PicoPure‱ RNA Isolation Kit from ThermoFisher Scientific following the protocol below:
Measure the RNA concentration using DS-11 FX+ Spectrophotometer Fluorometer and proceed for library construction of samples with input of 300ng.
rRNA depleted Hyperprep RNASeq Library Construction
The input parameters process workflow and library construction for the KAPA RNA HyperPrep Kit
for Illumina platforms is described below:
Indexes used are IDT for Illumina – TruSeq DNA UD Indexes v2 (96 Indexes, 96 Samples) #20040870
NovaSeq X Plus Workflow: 10B Flow Cell
The NovaSeq X Plus from Illumina is used for sequencing. Sequencing on this instrument is on a patterned flow cell with two color chemistry (green and blue). Sequencing is on a 10B Flow Cell with 8 lanes and can currently be done in 100 cycles, 200 cycles or 300 cycles. Note that each cycle kit comes with 38 cycles to account for index reads. Each lane yields 1250M reads.