Oct 20, 2023
Version 1
LDW_BDC Muscle scRNAseq V.1
- 1Cornell University
Protocol Citation: Lauren D Walter, Benjamin D Cosgrove 2023. LDW_BDC Muscle scRNAseq. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1q31ogr2/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: October 20, 2023
Last Modified: October 20, 2023
Protocol Integer ID: 89669
Abstract
single-cell RNA sequencing of muscle
Skeletal muscle injury and collection
Skeletal muscle injury and collection
In a given mouse, both tibialis anterior (TA) muscles were injected with 10 µl of notexin (10 µg/ml; Latoxan, France).
At various days post injury, the mouse was sacrificed and the TAs were collected. Each TA was proccessed independently to generate single cell suspensions.
Generate single-cell suspensions
Generate single-cell suspensions
Muscles were enzymatically digested with 8 mg/ml Collagenase D (Roche, Basel, Switzerland) and 10 U/ml Dispase II (Roche, Basel, Switzerland) and then manually dissociated to generate cell suspensions.
Myofiber debris was removed by filtering the cell suspensions through a 100 µm and then a 40 µm filter (Corning Cellgro # 431752 and # 431750).
After filtration, erythrocytes were removed by incubating the cell suspension in erythrocyte lysis buffer (IBI Scientific # 89135-030).
After digestion, the single-cell suspensions were washed and resuspended in 0.04% BSA in PBS at a concentration of 106 cells/ml. A hemocytometer was used to manually count the cells to determine the concentration of the suspension.
Single-cell RNA library preparation and sequencing
Single-cell RNA library preparation and sequencing
Single-cell RNA-sequencing libraries were prepared using the Chromium Single Cell 3’ reagent kit v3 (10x Genomics, Pleasanton, CA) following the manufacturer’s protocol.
Cells were diluted into the Chromium Single Cell A Chip to yield a recovery of 6,000 single-cell transcriptomes with <5% doublet rate.
Libraries were sequenced on the NextSeq 500 (Illumina, San Diego, CA).