title: GSM8833375: Memory B Cells, Gene Expression; Mus musculus; RNA-Seq
library_construction_protocol: Cells were first incubated with Fc receptor blockers anti-CD16/CD32 mAb and labelled with HEL at 200 ng/ml in PBA, after which cells were washed three times with PBA. Cells were then stained with anti-B220 BV786, anti-CD4 PE-Cy7, anti-Vα2 TCR FITC, anti-Vβ5.1/5.2 TCR PerCP-Cy5.5 mAbs, as well as TotalSeq-A (anti-CD19, CD38, CCR6, CD4, CD45.1, CD45.2) and hashtag oligonucleotide antibodies. Cells from different organs/conditions were stained with different TotalSeq-A hashtag antibodies: dLN (hashtag 1), ndLN (hashtag 2), spleen (hashtag 3), naïve LN (hashtag 4), and naïve spleen (hashtag 6). After staining with mAbs, cells were washed three times with PBA and finally resuspended in 500μl PBA. DAPI was added to each sample just before sorting at a final concentration 0.1μg/ml. HEL-binding B cells and Vα2+ Vβ5.1/2+ cells were sorted into RPMI 1640 with 10% FBS. The Garvan-Weizmann Centre for Cellular Genomics (GWCCG) performed the single-cell capture, library preparation, and sequencing as an in-house commercial service, using the Chromium Single-Cell v2 3' Kits (10x Genomics). The libraries were sequenced on Illumina NovaSeq 6000 (NovaSeq Control Software v1.7.0 / Real Time Analysis v3.4.4) using NovaSeq S2 130 cycles kit (Illumina, 20497313 as follows: 28 bp (Read 1), 91 bp (Read 2) and 8 bp (Index)). The sequencing of the libraries was performed by the Ramaciotti Centre for Genomics.
identifiers.namespace: GEO
identifiers.id: GSM8833375_r1