title: GSM4420017: B1: Bcell_aDEC_WT_LZ_GC_FUCCI; Mus musculus; RNA-Seq
library_construction_protocol: For bulk RNA sequencing experiments congenic OTII Tfh cells (CD4+, CD62low, CXCR5hi, PD-1hi) or positively selected B cells (500 cells) were purified by flow cytometry 18 hours after pre-immunized host mice were injected with aDEC-205 chimeric antibodies. 1ng of total RNA was used to generate full length cDNA using Clontech's SMART-Seq v4 Ultra Low Input RNA Kit The cDNA was used to prepare libraries using Illumina Nextera XT DNA sample preparation kit (Cat # FC-131-1024).   Libraries with unique barcodes were pooled at equal molar ratios and sequenced on an Illumina NextSeq 500 sequencer to produce 75bp reads, following manufactures protocol (Cat# 15048776). For single cell RNA sequencing, single cell spleen suspensions were prepared from half-spleens of NP-OVA immunized SellCreERT2 ROSAtdT mice on day 7 and 21 after immunization. Samples were indexed with TotalSeqC (BioLegend) cell surface antibodies and CD4+, CD62low, CD44hi, PD1hi, CXCR5high, tdTomato+ T cells were purified by flow cytometry, pooled and loaded on to a Chromium Controller (10x Genomics). Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 5′ v2 Reagent Kit (10x Genomics) according to manufacturer's protocol. Libraries were loaded onto an Illumina NextSeq with the mid-Output Kit (150 paired end) for V-D-J analysis or NOVAseq for single cell gene expression. Hashtag indexing was used to demultiplex the sequencing data and generate gene-barcode matrices, respectively.
attributes.tag: GEO Accession
attributes.value: GSM4420017