GSM8324293: Sample_STE_058_BT_Tcell; Homo sapiens; RNA-Seq
Extraction is included within the 10X genomics scRNA protocol Cellular suspensions were loaded on a Chromium controller (10x Genomics, Pleasanton, CA, USA) to generate single-cell Gel Bead-in-Emulsions (GEMs). Single-cell RNA-Seq libraries were prepared using Chromium Single-Cell 5′ v2 Library & Gel Bead Kit (PN-1000265, PN-1000264, 10x Genomics). The beads were dissolved and cells were lysed per manufacturer's recommendations. Briefly, the beads were dissolved, cells were lysed, and GEM reverse transcription (GEM-RT) was performed to produce barcoded, full-length cDNA. After incubation, GEMs were broken, the pooled post-GEM-RT reaction mixtures were recovered, and cDNA was purified with silane magnetic beads (DynaBeads MyOne Silane Beads, PN-37002D ThermoFisher Scientific, Waltham, MA). cDNA was amplified by PCR, generating sufficient material to construct B cell-enriched, T cell-enriched, and 5' gene expression libraries. The barcoded, full-length V(D)J segments were enriched by PCR with primers specific to the BCR and TCR constant regions using the Chromium Single Cell Human BCR Amplification kit (PN-1000253, 10x Genomics) and Chromium Single Cell Human TCR Amplification kit (PN-1000252, 10x Genomics), respectively. cDNA library amplicon size was optimized by enzymatic fragmentation and size selection. 5' gene expression, BCR, and TCR indexed sequencing libraries were constructed by End Repair, A-tailing, Adaptor Ligation, and PCR. Final libraries contain the P5 and P7 priming sites used in Illumina bridge amplification. The 5' gene expression, BCR, and TCR libraries were sequenced following 10X Genomics read length and sequencing depth guidelines on the NovaSeq6000 sequencer (Illumina).