title: Whole Genome Sequencing of splenocytes from Four Core Genotype (FCG) mice
design: Whole Genome 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)8. 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. Wild-type C57BL/6J males (Janvier-Labs) were imported and housed for one week prior to sacrifice. 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 unless otherwise stated. Whole livers were dissected out, transferred to 5ml tubes, snap-frozen on liquid nitrogen and stored at -80˚C. Frozen livers were pulverised with the cryoPREP® Automated Dry Pulverizer (Covaris #CP02) using the TT2 holder, TT2 consumables and impact setting 6 and pulverised material stored at -80˚C. DNA was isolated from ~30 mg frozen liver and 2mm3 ear punch biopsies using a protocol incorporating elements of the AllPrep DNA/RNA Micro Kit (Qiagen) and a DNeasy Blood and Tissue Kit (Qiagen). Frozen tissue samples were homogenised by adding 600 uL RLT containing 1% β-Mercaptoethanol and a 5mm stainless steel bead (Qiagen # 69989) and processing for 40 seconds at 15 Hz with the TissueLyser II. Lysed samples were supplemented with 20ul proteinase K (from the Blood and Tissue Kit) and incubated at 56°C for 10 min. Half of the digested sample was combined with 500uL Buffer AL (Qiagen Blood & Tissue), mixed by vortexing and incubated at 56°C and 500 rpm for 30 min. An equal volume of absolute ethanol was added to the samples and mixed thoroughly. The precipitated mixture was transferred to a DNeasy Mini column and washed twice with 500 μl buffer AW1 and subsequently with 500 μl AW2. After drying the membrane for 2 minutes at 20,000 x g, DNA was eluted using 50 μl AE buffer pre-warmed to 70°C. DNA quality and quantity were assessed using a Genomic DNA ScreenTape (Agilent Technologies) and Qubit dsDNA BR Assay Kit (Life Technologies). Dual Index whole genome libraries were prepared with 300ng of genomic DNA using the NEBNext Ultra II kit (E7805S/L). Fragmentation time was optimised and occurred at 37°C for 20min. Adaptor-ligated DNA was size selected with AMPure XP Beads (Beckman Coulter) to select an insert size of approximately 150-200bp. Libraries were amplified 6 cycles using the NEBNext UDI primers (E6440). Library size and molarity were determined using a D5000 ScreenTape (Agilent Technologies) and Qubit dsDNA HS Assay Kit (Life Technologies). Final library size was approximately 600-700bp.