title: Single-cell RNA-Sequencing of splenocytes from Four Core Genotype (FCG) mice
design: Single-cell RNA-Sequencing of splenocytes from Four Core Genotype (FCG) mice
library_construction_protocol: Four Core Genotypes colony founders on the C57BL/6J background were obtained from The Jackson Laboratory (strain 010905). Two XYT individuals were mated with wild-type C57BL/6J (Janvier-Labs) or CAST/EiJ (bred at the German Cancer Research Center) females to respectively generate pure C57BL/6J and B6 x CAST F1 hybrid XXO, XXT, XYO and XYT progeny. All animals were maintained as virgins (apart from the two XYT founders) and housed in groups of up to six mice in Tecniplast GM500 IVC cages with a 12 hour light/dark cycle. Mice had ad libitum access to water, food (Kliba 3437) and environmental enrichment. The colony was regularly controlled for infections using sentinel mice to ensure a healthy status. All breedings and organ collections were carried out with the approval of the Regierungspräsidium Karlsruhe. Animal genotype (i.e. XXO, XXT, XYO or XYT) was assigned based on independent assessment of sex chromosome complement and gonadal sex. Sex chromosome complement was initially determined by PCR detection of the Y chromosome using published primers YMTFP1 (5' CTG GAG CTC TAC AGT GAT GA 3') and YMTRP1 (5' CAG  TTA  CCA  ATC  AAC  ACA  TCA  3')18 and confirmed by detection of Xist (specific to the XX genotypes) and Sry (specific to the XY genotypes) sequencing signal. Gonadal sex was determined by visual assessment of external morphology, initially at the time of weaning and confirmed at the time of sacrifice. Animals were sacrificed by carbon dioxide inhalation at 12 weeks of age. Whole spleens were dissected, transferred to a petri dish containing DMEM (Gibco # 41966029) with 5% fetal bovine serum (FBS, Thermo Fisher # 16140071) and processed directly for single cell isolation. All tissue collections were performed between 8:00 and 12:00 to approximately control for circadian regulation of organ physiology. Dissected spleen fragments were transferred to a 40-μm cell strainer (Greiner # 542040) and ground with a syringe plunger. The cell suspension was strained twice. Strainers were washed with additional DMEM (Gibco # 41966029) with 5% FBS and cells were pelleted by centrifugation at 300 x g for 4 minutes at 4°C. Cell pellets were carefully resuspended in 300 uL of ACK lysis buffer (Lonza # BP10-548E) for 1 minute in ice. After incubation cells were washed with 1mL of DMEM with 5% FBS and pelleted. Cells concentration was determined using 0.4% trypan blue stain (Invitrogen) and Countess II automated cell counter (Invitrogen). 1 × 10^7 cells were processed for dead cell depletion using Dead Cell Removal Kit (Miltenyi Biotec # 130-090-101) as per the manufacturer's instructions. The magnetically labelled dead cells were retained using MS MACS Columns (Miltenyi Biotec # 130-042-201) on a MiniMACS Separator (Miltenyi Biotec # 130-042-102). The live cell fraction was pelleted and resuspended in 1X PBS with 0.04% bovine serum albumin (BSA, Miltenyi Biotec # 130-091-376) to a final concentration of 1 × 106 cells/mL (1,000 cells per μL).  Single-cell RNA was performed with 20.000 cells which were encapsulated into droplets in the Chromium Controller instrument using Chromium Next GEM Single Cell 3' Reagents Kits v3.1, according to the manufacturer's recommendations. Briefly, single cell lysis and barcoded reverse transcription (RT) of mRNA happened into the droplets. Twelve cycles were used for cDNA amplification and sample index PCR to generate Illumina libraries.