GSM6215440
Samples were prepared using the General Sample Preparation protocol from 10× Genomics (10×, Manual Part #CG00053) adapted from published methods Cells are combined with a master mix that contains reverse transcription reagents. The gel beads carrying the Illumina TruSeq Read 1 sequencing primer, a 16bp 10x barcode, a 12bp unique molecular identifier (UMI) and a poly-dT primer are loaded onto the chip, together with oil for the emulsion reaction. The Chromium Controller partitions the cells into nanoliter-scale gel beads in emulsion (GEMS) within which reverse-transcription occurs. All cDNAs within a GEM, i.e. from one cell, share a common barcode. After the RT reaction, the GEMs are broken and the full length cDNAs cleaned with Silane Dynabeads. cDNAs are then amplified and purified by SPRI beads. After purification, the cDNAs are assayed on an Agilent 4200 TapeStation High Sensitivity D5000 ScreenTape (Santa Clara, CA) for qualitative and quantitative analysis. Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size.  Illumina  P5 and P7 sequences (San Diego), a sample index, and TruSeq read 2 primer sequence are added via End Repair, A-tailing, Adaptor Ligation, and PCR.  The final libraries contain P5 and P7 primers used in Illumina bridge amplification. Sequence is generated 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 an Illumina sequencing platform at a minimum of 50k reads/cell