title: GSM3746212: Muscle_1; Homo sapiens; RNA-Seq
library_construction_protocol: Four individual samples of dissociated cryopreserved cells (non-fiber cells only) obtained from a single human muscle biopsy were thawed based on the 10X Genomics Demonstrated Protocol (Manual Part Number CG00039 Rev C). Briefly, cryovials were removed from liquid nitrogen storage and immediately thawed in a 37°C waterbath for 2 minutes. 1ml of the thawed cells were transferred into individual tubes, the cryovials were rinsed with 1ml of warm complete growth medium and the rinse medium was added dropwise (1 drop per 5 sec) to each tube containing the thawed cells. The cells were serially diluted with complete growth medium a total of 3 times by 1:1 volume dropwise additions with 1 minute wait before each addition. Next the cells were centrifuged at 300 rcf for 5 minutes and resuspended in complete growth medium. Cells were filtered through a 40μm cell strainer, counted, and the volume of medium was adjusted for the final concentration to be 1,000 cells/μl 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(first 16 nt) + UMI(next 10 nt)), 8 bp i7 index (sample index), and 98 bp Read2 (insert).
attributes.tag: GEO Accession
attributes.value: GSM3746212