GSM5322429: Memory B cells from Lung; Mus musculus; RNA-Seq
10x 5' scRNA-seq libraries were prepared according to the manufacturer's instructions with modifications for generating the BCR-seq libraries. Following cDNA amplification, SPRI select beads were used to separate the large cDNA fraction derived from cellular mRNAs (retained on beads) from the HTO-containing fraction (in supernatant). For the cDNA fraction derived from mRNAs, 50ng were used to generate transcriptome library and around 5ng were used for BCR library construction. Gene expression libraries were prepared according to manufacturer's instructions. For BCR libraries, heavy and light chain cDNA were amplified by two rounds of PCR (6 cycles + 8 cycles) using external primers recommended by 10x Genomics, and 800 pg of purified amplified cDNA was tagmented using Nextera XT DNA sample Preparation kit (Illumina) and amplified for 12 cycles using the SI-PCR forward primer (10x Genomics) and a Nextera i7 reverse primer (Illumina). For the HTO-containing fraction, 5ng were used to generate the HTO library. The resulting libraries were pooled and sequenced together on an Illumina NextSeq550 platform, using High Output 75-cycle flow cells, targeting 5×104 reads per cell for gene expression, 5x103 reads per cell for BCR, 2x103 reads per cell for hashtag, in paired-end single-index mode (Read 1: 26 cycles, Read i7: 8 cycles, Read 2: 57 cycles).  FB5P-seq: For each plate, library preparation consisted in RT with template switching for incorporating well-specific barcodes and UMIs, cDNA amplification with 22 cycles of PCR, pooling of 96 wells into one tube, and 5'-end RNA-seq library preparation using tagmentation-based modified Nextera XT DNA sample Preparation kit (Illumina). Libraries were tagged with a plate-specific i7 index and were pooled for sequencing on an Illumina NextSeq2000 platform, with P2 flow cells, targeting 2.5×105 reads per cell in paired-end single-index mode (Read 1: 103 cycles, Read i7: 8 cycles, Read 2: 16 cycles).