GSM4230228: muscle_P; Mus musculus; RNA-Seq
Skeletal muscle single-cell suspension was prepared by collagenase/dispase digestion. Briefly, each tissue was minced in 2.5 ml digestion solution (1 U/ml collagenase B and 1 U/ml dispase II (Roche Diagnostics, Indianapolis, IN, USA) in PBS) and incubated at 37ºC for 1 hour. The reaction was terminated by adding 10 ml PBS containing 10% fetal bovine serum (FBS). The mixture was then filtered through a 70-um cell strainer and centrifuged at 250 g for 5 minutes. The pellet was collected and the supernatant was centrifuged again at 250 g for 5 minutes. The pellet was combined with the pellet from the first centrifugation, washed with PBS, and centrifuged at 670 g for 10 minutes. The pellet was re-suspended in 3 ml PBS and filtered through a 40-um cell strainer. Cell suspension was layered on equal volume of the Lympholyte-M solution (Cedarlane, Burlington, NC, USA) and centrifuged at 2,095 g for 45 minutes. Cells at the interface were collected, centrifuged at 670 g for 10 minutes, and re-suspended in FACS staining buffer. Resident macrophages were sorted as CD45+CD11b+F4/80+CD64+ cells. Single-cell encapsulation with beads using the 10X Genomics Chromium Single Cell 3' v2 kit (PN 120235 and PN 120234 Module 1) was performed following manufacturer instructions (User Guide Rev A, 10X Genomics, Pleasanton, CA, USA). Briefly, 4 wells of a 10X microfluidic chip (PN 120236) were loaded each with an individual sample to target 5,000 cells per sample. Single-cell gel beads in emulsion (GEMs) were generated and reverse-transcription was performed in the emulsion prior to 12 cycles of cDNA amplification. Quality control and quantification of the amplified cDNA were assessed by Bioanalyzer. Libraries were constructed according to manufacturer instructions (PN 120234 Module 2). Each library was tagged with a different index for multiplexing (PN 120262) during sequencing. Library quality controls were assessed by Bioanalyzer and quantified by Qubit and quantitative PCR (KAPA Biosystems Quantification for Illumina platforms). Sequencing was performed on an HiSeq 2500 (Illumina, Inc., San Diego, CA) using the following read length: 26 bp Read1 (cell barcode + UMI), 8 bp i7 index (sample index), and 98 bp Read2 (insert).
GEO Accession
GSM4230228