GSM5331704: F_WT_ABC_2; Mus musculus; RNA-Seq
RNAseq: Total RNA from sorted B -lymphocytes was isolated using the RNeasy Plus Mini kit (Qiagen). ATACseq: The nuclei of sorted ABCs from female DKO, male DKO, or Yaa-DKO mice were prepared by incubation of cells with nuclear preparation buffer (0.30M sucrose, 10mM Tris pH 7.5, 60mM KCl, 15mM NaCl, 5mM MgCl2, 0.1mM EGTA, 0.1% NP40, 0.15mM spermine, 0.5mM spermidine, and 2mM 6AA) Cut&Run: Sorted cells were immediately incubated overnight with H3K27me3 (anti- Tri-Methyl-Histone H3 (Lys27) (Cell Signaling Technology #Rabbit mAb #9733) or IgG isotype controls (Guinea Pig anti-Rabbit IgG (H+L) Secondary Antibody (novusbio #NBP1-72763) at 1:50 dilution of antibody binding buffer with a final concentration of 0.05% digitonin. Following incubation and digestion CUTANA™ pAG-MNase from EpiCypher (SKU: 15-1116) the digested DNA fragments were release and purified by QIAquick PCR purification kit (#2810). RNAseq: Quality of all RNA and library preparations were evaluated with BioAnalyzer 2100 (Agilent). Sequencing libraries were sequenced by the Epigenomics Core Facility at Weill Cornell Medicine using a HiSeq2500, 50-bp paired-end reads at a depth of ~15-50 million reads per sample ATACseq: sequencing libraries were constructed as described in PMID: 26083756: Buenrostro JD, Wu B, Litzenburger UM, Ruff D, Gonzales ML, Snyder MP, Chang HY, Greenleaf WJ. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature. 2015 Jul 23;523(7561):486-90. doi:10.1038/nature14590. Epub 2015 Jun 17. Cut&Run:The resulting DNA fragments were end-repaired and barcoded libraries were made with the NEB Ultra II DNA Library Prep kit ((E7645S, E7335S & E7500S)). The libraries were sequenced on Illumina NextSeq 500 to a minimum of 15 million reads per sample.