GSM7567490: B16 Control; Mus musculus; RNA-Seq
Each prep was titered to contain 10,000 cells per library construction. Cells were combined with master mix containing reverse transcription (RT) reagents. Then the cell RT reaction mix, gel beads carrying the Illumina TruSeq Read 1 sequencing primer, a 16 bp 10x barcode, a 12 bp unique molecular identifier (UMI), and a poly-dT primer together with oil for the emulsion reaction were loaded onto the 10x Chromium Chip G. 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, shared a common barcode. After the RT reaction, GEMs were broken and full length cDNAs were cleaned with Silane Dynabeads. cDNAs were then amplified and purified using SPRI beads. After purification, cDNAs were assayed on an Agilent 4200 TapeStation High Sensitivity D5000 ScreenTape (Agilent Technologies, Santa Clara, CA) for qualitative and quantitative analysis. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. Illumina P5 and P7 sequences (Illumina, San Diego, CA), a sample index, and TruSeq read 2 primer sequence were added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contained P5 and P7 primers used in Illumina bridge amplification. Libraries were constructed at the Molecular Genomics Core of Duke University. Libraries were prepared following the 10x Genomics Single Cell 3' v3.1 protocol (10x Genomics, Pleasanton, CA).