Nov 14, 2024
Sample preparation for Single cell RNA sequencing (scRNA-seq)
- Jun B. Ding1,
- Richard H. Roth1,
- Fuu-Jiun Hwang1,
- Yue Sun1
- 1Stanford University
Protocol Citation: Jun B. Ding, Richard H. Roth, Fuu-Jiun Hwang, Yue Sun 2024. Sample preparation for Single cell RNA sequencing (scRNA-seq). protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpz1wjlzp/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: August 29, 2024
Last Modified: November 14, 2024
Protocol Integer ID: 106637
Keywords: ASAPCRN, RNA, Sequencing, scRNA-seq, Brain
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020551
Abstract
Mouse brain tissue sample preparation for single cell RNA sequencing (scRNA-seq) that is performing in the Ding lab.
Attachments
Safety warnings
Wear appropriate PPE as required by your institution.
Ethics statement
Prior ethics approval (e.g. IACUC) should be obtained before performing these experiments. Approval was obtained by the Stanford University IACUC before any procedures were performed.
Solution Preparation (in advance)
Solution Preparation (in advance)
Solutions recipes with catalog information can be found in attached document
- ACSF 10X
- HBSS
- Isethionate Solution
- Kynurenic Acid Solution
- Nitro-Arginine Solution
- Glutathione Solution
- Sodium Pyruvate Solution
- Albumin Inhibitor (Worthington Cat# LS000290): Add 32 mL of HBSS to the albumin bottle and allow it to dissolve at room temperature. Mix by inversion, aliquot 3 mL per tube, and store at 4 °C .
Solution Preparation (day of processing)
Solution Preparation (day of processing)
Solutions recipes with catalog information can be found in attached document
- 1x ACSF: Check osmolality (~300 mOsm). Bubbling with 95/5 O2/CO2
- HBSS with supplements (HBSS/s). Bubbling with pure O2. Isethionate Solution with supplements (Ise/s). Bubbling with pure O2.
- Papain (Worthington LK003176) solution: adding 5 ml oxygenated HBSS/s to the bottle and incubating at 37°C. Transfer the Papain solution into spin flask (Wheaton #3572651). Displace the pure O2 tube in the flask, but do not bubble gas through the solution. Check the osmolality every 20 min (~ 320 mOsm).
- Sample buffer: 1X PBS (calcium and magnesium-free) containing 0.04% BSA (400 µg/ml)
Cutting acute slices
Cutting acute slices
Perfuse the mouse with 25 ml of cold, freshly prepared, oxygenated 1x ACSF and dissect out the brain.
Cut the brain using a PFA-free vibratome (thickness 300 um, speed 1.0 mm/s, amplitude 1.4 mm).
Transfer slices to a chamber with bubbling ACSF at 34 °C and incubate for
00:30:00 .
30m
Micro-Dissection
Micro-Dissection
Dissect the desired region(s) of interest/tissue in a plate containing oxygenated ACSF.
Refresh the solution if the dissection takes more than 00:03:00 .
3m
Transfer the tissue pieces to HBSS/s bubbling with pure O2.
Papain incubation
Papain incubation
1h
1h
Transfer the tissue to a spin flask with Papain solution and allow it to settle at the bottom.
Place the O2 tube on the surface of the solution.
Incubate at 37 °C with slow, constant agitation for 01:00:00 using a use a spinner flask (WHEATON #356943) on a magnetic stirrer. The setup looks like this:
1h
Trituration
Trituration
Transfer the tissue to a 15 mL tube and centrifuge at 300 x g for 00:05:00 .
5m
Discard the supernatant and add 5 ml HBSS/Proteinase Inhibitor.
Triturate the tissue using fine-polished Pasteur pipettes (~600 µm, 10 times; ~300 µm, 10 times; ~100 µm, 5 times).
Filter through a 40 µm cell strainer (Falcon Facs tube #352235) and centrifuge at 300 x g for 00:05:00 . Avoid making bubbles.
5m
Debris Removal
Debris Removal
6m
6m
Discard the supernatant and resuspend the cells in 500 uL sample buffer.
Layer the cloudy cell suspension on 5 mL albumin and centrifuge at 70 x g for 00:06:00
Note: A benchtop centrifuge without the brake setting was used and allowed to completely stop; with the very slow spin-down, the gradient should remain undisturbed even with sudden braking.
6m
Discard the supernatant and wash once with 500 uL sample buffer.
Dead Cell Removal
Dead Cell Removal
Follow the protocol for Annexin V-conjugated magnetic beads (Miltenyi Biotec, Cat# 130-090-201).
Resuspend the cells in sample buffer (~50uL -100uL).
Check viability and density under a microscope and place the cells on ice.
Cell Quality for library prep
Cell Quality for library prep
For prepare single cell library with Chromium (10x Genomics), cell quality should be:
Viability > 70%
700~1200 cells/ml
50~ 100 ul volume