GSM9618412: 10xHTO_batch2; Homo sapiens; RNA-Seq
RNA was extracted from single B cells using RNA clean XP beads (Beckman Coulter) and 5' RACE was performed to synthesize and amplify cDNA. A second round of PCR was performed to amplify DNA for IgG, IgA, IgK, and IgL sequences. SmartSeq: Ig enrichment products were prepared into DNA libraries using the Illumina Nextera XT library preparation kit (Illumina). The Illumina-ready libraries were sequenced on a Nextseq 2000 (Illumina) using a P1 300 cycle kit (Illumina) for paired end 2x150 base pair reads to obtain V(D)J sequences;10x:To analyze the V(D)J repertoires sorted single B cells, a cell suspension was loaded on the 10x Genomics Chromium Instrument with reagents from the Next GEM Single Cell 5' Kit v2 (10x Genomics P/N 1000263) to generate gel bead-in emulsions. Following the manufacturer's protocol, GEM-RT was performed followed by purification and amplification of the total cDNA. Heavy and light chains were amplified using Ig-specific 3' primers with the addition of Illumina sequences. The Illumina-ready libraries were sequenced on a Nextseq 2000 (Illumina) using a P1 600 cycle kit for paired end 2x 300 base pair reads. For hashing data, the 10x Genomics protocol for sequencing of surface DNA was followed.