GSM5693859: ATAC MPP3 Ebf1-KO rep 1; Mus musculus; ATAC-seq
Mouse BM cells were isolated from pooled femora, tibiae and ilia bones, by crushing them in cold-PBS-3%FCS. For progenitor cell sorting, bone marrow cells were incubated with a biotin-antibody cocktail (CD3, B220, CD11b, Gr1, Ter119), followed by magnetic depletion with streptavidin-conjugated magnetic nanobeads (BioLegend). MPP3 (LSK+ CD150- CD48+) and MPP4 (LSK+ CD150- CD48+ Flt3+) cells were sorted by a FACS Aria III or FACSymphony instrument (BD Biosciences). MPP3 and MPP4 cell pellets were resuspended in a lysis/transposition buffer (1 µl Tagmentase, 10 µl 2x TD buffer (Diagenode), 0.01% digitonin (Promega), 0.3x PBS) and incubated for 30 min at 37°C at 700 rpm. Reactions were cleaned up with Zymo DNA Clean and Concentrator -5 columns (Zymo Research). ATAC-sequencing libraries were prepared as described in Buenrostro et al. 2015, using Nextera DNA UD Indexes (IDT); 10-12 PCR amplification cycles and double-sided size selection was performed. Paired-end 100 bp reads were generated using the Illumina NovaSeq 6000 system. Samples were first sequenced at shallow depth (~5 million reads) to assess the quality, prior to sequencing at a depth of 50 million reads. Reads from both flowcells were merged at the fastq level.