title: GSM7360914: Combined scRNAseq of 3 LDHA-sufficient and 3 LDHA-deficient pre-GC and GC B cells; Mus musculus; RNA-Seq
library_construction_protocol: Spleens from three LDHA-sufficient mice and three LDHA-deficient mice were isolated into single cell suspensions. Cells were then stained with standard flow cytometry antibodies, as well as hashtag B2m and CD45 antibodies to barcode each mouse with a unique Oligonucleotide Hash Tag. Cells were sorted for viability and B cell-positivity, as well as based on GL7 expression (see S5c). Purified Hash-tagged cells were counted and resuspended at a concentration of 1000 cells/ul. Cells suspension was  verified to have viability of >80% using a Countess II. Cells were captured in droplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken and cDNA purified using Dynabeads MyOne SILANE followed by PCR amplification per manual instruction. Approximately 30,000 cells were targeted, where several samples were multiplexed together on one lane of 10X Chromium, following cell hashing protocol. Library was prepared using Chromium Single Cell 5ʹ v2 protocol (10x Genomics). The scRNA-seq and scBCR-seq libraries were prepared using the 10x Single Cell Immune Profiling Solution Kit, according to the manufacturer's instructions. Briefly, amplified cDNA was used for both 5′ gene expression library construction and TCR enrichment. For gene expression library construction, amplified cDNA was fragmented and end-repaired, double-sided size-selected with SPRIselect beads, PCR-amplified with sample indexing primers (98 °C for 45 s; 14–16 cycles of 98 °C for 20 s, 54 °C for 30 s, 72 °C for 20 s; 72 °C for 1 min), and double-sided size-selected with SPRIselect beads. For BCR library construction, BCR transcripts were enriched from 2 μl of amplified cDNA by PCR (primer sets 1 and 2: 98 °C for 45 s; 10 cycles of 98 °C for 20 s, 67 °C for 30 s, 72 °C for 1 min; 72 °C for 1 min). Following BCR enrichment, enriched PCR product was fragmented and end-repaired, size-selected with SPRIselect beads, PCR-amplified with sample-indexing primers (98 °C for 45 s; 9 cycles of 98 °C for 20 s, 54 °C for 30 s, 72 °C for 20 s; 72 °C for 1 min), and size-selected with SPRIselect beads. Final libraries (GEX, TCR and HTO) were sequenced on Illumina NovaSeq S4 platform (R1 – 26 cycles, i7 – 10 cycles, i5 – 10 cycles, R2 – 90 cycles).