6 protocols
nucleic acid sequencing protocol
Purified DNA was eluted in 20 µl Buffer EB (QIAGEN) and sequenced using paired-end sequencing on a NextSeq 550 instrument (Illumina).
nucleic acid extraction protocol
Supernatant was removed and 50 µl of transposase mixture (25 µl TD buffer (Illumina), 2.5 µl of TDE1 (Illumina), 0.5 µl of 1% digitonin (Promega), 22 µl nuclease-free water) added to the cells. Cells were then incubated at 37°C for 30 min at 300 rpm in an Eppendorf ThermoMixer.
sample collection protocol
Fast-ATAC sequencing was performed as previously described (Corces et al. 2016). Briefly, 5,000 viable PbTII cells were sorted by flow cytometry and pelleted by centrifugation.
nucleic acid library construction protocol
Transposed DNA was amplified and purified using a QIAGEN MinElute PCR Purification kit, according to the manufacturer’s protocol.
normalization data transformation protocol
Unmapped reads, reads mapping to unassigned contigs and mate unmapped reads were removed, as well as mitochondrial genes and PCR duplicates. The resulting .bam files were first converted to bedGraph using the bedtools genomecov command from the BEDTools suite and then converted to bigwig format using the bedGraphToBigWig program from University of California, Santa Cruz (UCSC) Genome Browser (https://genome.ucsc.edu/). Read counts were normalised to the number of uniquely mapped reads per million.
high throughput sequence alignment protocol
Raw ATAC-seq reads were mapped to mouse genome MGSCv37 (mm9) using BWA-MEM.