Apr 17, 2026
Human CSF Processing for 10x scRNA-seq
- 1Yale University
Protocol Citation: Le Zhang 2026. Human CSF Processing for 10x scRNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyn63rvx9/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: March 28, 2022
Last Modified: April 29, 2026
Protocol Integer ID: 59971
Keywords: 10x CSF Single Cell, CSF Cells Freezing, Cerebrospinal Fluid (CSF), ASAPCRN, team hafler csf processing protocol for 10x, team hafler csf processing protocol, single cell rna, human csf processing
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000529
Abstract
This protocol details about team Hafler CSF processing protocol for 10x single cell RNA sequencing.
Attachments
392-855.docx
17KB
Materials
Materials
- 10x Genomics Chromium Single Cell 5' Reagent Kit v2 chemistry
- Bambanker cell freezing medium
- Sprotte atraumatic lumbar needle with cannula
- CSF WASTE tube
- Swing bucket centrifuge
- C-Chip disposable hemacytometer (DHCN015)
- Mr. Frosty freezing container
CSF Collection
Collect the cerebrospinal Fluid (CSF) Room temperature (64-77°F / 18-25°C). No fasting is required. Once collected, keep samples at On ice or at 4 °C .
Perform lumbar puncture (LP) utilizing Sprotte atraumatic lumbar needle with cannula.
Note
In case of bleeding at the puncture site, collect the first 1-2 mL of CSF fluid in the CSF WASTE tube.
Continue collecting CSF fluid in the CSF WASTE tube until CSF is visibly devoid of red blood cells (RBC) contamination, i.e., no pinkish or red color. For the CSF sample, collect 30 mL of CSF fluid into pre-labeled tubes. Proceed with the next steps even if less than 30 mL of CSF was collected. Replace caps after CSF sample collection and mix gently by inverting each tube 3-4 times. CSF WASTE tube may be discarded.
Record the total CSF volume and if CSF sample looks grossly or visibly contaminated with RBC and/or if it is hued pink or red. Record the date and time of the CSF sample collected.
Transfer the CSF sample as quickly as possible to the lab. Then proceed to CSF centrifugation and processing.
10x CSF Single Cell Protocol
Spin down the CSF cells at 300 x g, 4°C, 00:10:00 using a swing bucket centrifuge.
10m
Aspirate the supernatant with a pipette, leaving ~500 µL without disturbing the cell pellet. Save the CSF supernatant On ice for further aliquots.
Mix and count the total CSF cell number. Use 5 µL of the sample with 1:1 dilution of trypan blue and a C-Chip disposable hemacytometer (DHCN015). Record the total CSF cell number.
Transfer to a 1.5-mL Eppendorf tube and spin down at 300 x g, 4°C, 00:10:00 using a swing bucket centrifuge.
10m
Carefully aspirate the supernatant, leaving ~50 µL .
Resuspend the cells and count.
If the cells need to be diluted more, use the CSF supernatant.
If there are leftover CSF cells after 10x loading, freeze down the cells.
Submit for 10x single cell loading. Ideally 700-1200 cells/ul. If too few cells, submit all the sample of 50 µL .
Use 10x Genomics Chromium Single Cell 5' Reagent Kit v2 chemistry for single cell gene expression and TCR/BCR sequencing.
CSF Cells Freezing Protocol
5m
Spin down the CSF cells at 400 x g, 4°C, 00:05:00 using a swing bucket centrifuge.
5m
Carefully aspirate the supernatant.
Resuspend the cell pellet in 0.1 mL of Bambanker cell freezing medium.
Freeze in Mr. Frosty freezing container in -80 °C freezer. Transfer to liquid N2 tank after 24h - 72h.
Transfer to liquid N2 tank after 24h - 72h.