GSM9006160: uninjured TA; Mus musculus; RNA-Seq
Muscles were dissected, minced, digested, strained and washed to isolate a single cell suspension Libraries are prepared from single cell suspensions isolated from tissue according to the 10x Genomics 3' scRNAseq user guide CG000204 (Pleasanton, CA). Cells are combined reverse transcription (RT) reagents and loaded onto a microfluidics chip along with individually barcoded gel beads and oil to partition each sample into nanoliter-scale gel beads in emulsion (GEMS), within which the RT reaction occurs. Next, the GEMs are broken to reveal full length cDNAs that are then purified, amplified, and enzymatically fragmented before Illumina P5 and P7 sequences (San Diego, CA), a sample index, and TruSeq read 2 primer sequence are added via End Repair, A-tailing, Adaptor Ligation, and PCR.  The cDNA and final libraries are checked for quality on the Agilent 4200 TapeStation System (Santa Clara, CA) quantified using the KAPA Library Quantification Kit (Roche – Indianapolis, IN). Libraries are sequenced using paired end sequencing (one end to generate cell specific, barcoded sequence and the other to generate sequence of the expressed poly-A tailed mRNA) on the Illumina sequencing platform at a minimum of 50k reads/cell.