GSM6360706: ATACseq_DKO; Mus musculus; ATAC-seq
Bilateral femurs and tibias were harvested from two wildtype C57BL/6 mice. Bones were flushed with 1X PBS with 2% fetal bovine serum (FBS), 0.1% sodium azide and 0.5 mM ethylenediaminetetraacetic acids, pH 7.4. The cells were filtered through a 70 µm mesh, centrifuged at 1200 rpm for 5 minutes and then incubated for 5 minutes with 5 mL of ACK lysis buffer for red blood cell lysis. Cells were then washed and centrifuged at 1200 rpm and subsequently resuspended. Cells were stained with two distinct oligo-labeled antibodies that recognize CD45 and MHC class I, which allow identification of cells derived from each individual mouse (referred to as hashtag antibodies).  We further stained cells with a panel of CITE-Seq antibodies (B220, CD19, CD93, CD25, IgM, and CD43) as well as fluorescently labeled B220 and CD43. Cells were sorted at a 1:1 ratio of B220+CD43+ and B220+CD43- cells to enrich for early progenitor B cell subsets. For 10X Genomics scRNA-seq, we generated three libraries that measure: (1) mRNA transcript expression (RNA), (2) mouse-specific hashtag oligos (HTO), and (3) cell surface marker levels using antibody derived tags (ADT). Cells were harvested and stained as described above. In order to have balanced populations of various B cell development stages, we enriched for earlier progenitor B cells (B220+CD43+) by sorting at a 1:1 ratio of Dump-B220+CD43+ cells and Dump-B220+CD43- cells. A total of 20,000 cells per mouse were sorted into a single microtube containing 1X PBS with 50% FBS and were washed and resuspended in 1X PBS with 10% FBS prior to cell capture. The sample was split into three libraries (RNA, HTO, and ADT). Reverse transcription PCR and library preparation were carried out under the Chromium Single Cell 3' v3 protocol (10X Genomics) per manufacturer's recommendations. After library prep, quality control was performed using a bioanalyzer (Agilent 2100 Bioanalyzer; Agilent Technologies) and preliminary sequencing of the RNA library on a MiSeq (Illumina) to determine the approximate number of cells and general quality. After passing quality control, the library was sequenced on the NovaSeq 6000 with 2x150 bp paired-end reads (Illumina). For single-cell ATAC-sequencing, we harvested bone marrow from wildtype (n = 1), Ncor1 KO (n = 1) and Ncor1/2 DKO (n = 1). Cell sorting scheme was identical as the single-cell RNA-seq cell sorting scheme, except we sorted 50,000 B220+CD43+ cells and 50,000 B220+CD43- cells, for a total of 100,000 cells for each genotype. Sorted cells were centrifuged at 300 x g for 5 minutes at 4Co. Cells were then resuspended in 100 µl of chilled lysis buffer, which consists of 10mM Tris-HCl pH 7.4 (T2914, Sigma-Aldrich), 10mM NaCl (S5150, Sigma), 3mM MgCl2 (M1028, Sigma-Aldrich), 0.1% Tween 20 (BP337, Fisher Scientific), 0.1% nonidet P40 substitute (74385, Sigma-Aldrich), 0.01% digitonin (BN2006, ThermoFisher), 1% BSA (130-091-376, Miltenyi Biotec), and nuclease-free water, and incubated on ice for 5 minutes. After incubation, 1 mL of chilled wash buffer, including 10mM Tris-HCl pH 7.4 (T2914, Sigma-Aldrich), 10mM NaCl (S5150, Sigma), 3mM MgCl2 (M1028, Sigma-Aldrich), 0.1% Tween 20 (BP337, Fisher Scientific), 1% BSA (130-091-376, Miltenyi Biotec), and nuclease-free water, was added to lysed cells, and centrifuged at 500 x g for 5 minutes at 4Co. Cells were resuspended in diluted nuclei buffer and nuclei permeabilization was determined using a Countess II (ThermoFisher). Cells were then captured on the 10X Chromium and subsequent library prep was generated using the Chromium Next GEM Single Cell ATAC library kit v1.1 (PN-1000164, 10X Genomics), following the protocol from manufacture recommendations. Post-library quality control (QC) was performed for both sizing and quantification using the Agilent Bioanalyzer (Agilent 2100 Bioanalyzer; Agilent Technologies) and KAPA library quantification kit for Illumina platforms (KR0405, Roche). All libraries were then sequenced on a NovaSeq 6000 with 2x150 bp paired-end reads (Illumina)