GSM4395722: Liver_Fibroblasts_SteadyState_ATAC_2; Mus musculus; ATAC-seq
Organs were digested with Accumax and structural cells isolated using FACS Chromatin accessibility mapping by ATAC-seq was performed as previously described (Buenrostro et al., 2013; Corces et al., 2016), with minor adaptations. In each experiment, a maximum of 50,000 sort-purified cells were collected at 300 g for 5 min at 4 °C. After centrifugation, the pellet was carefully resuspended in the transposase reaction mix (12.5 µl 2xTD buffer, 2 µl TDE1 (Illumina), 10.25 µl nuclease-free water, and 0.25 µl 1% digitonin (Promega)) for 30 min at 37 °C. Following DNA purification with the MinElute kit eluting in 11 µl, 1 µl of the eluted DNA was used in a quantitative PCR reaction to estimate the optimum number of amplification cycles. The remaining 10 μl of each library were amplified for the number of cycles corre-sponding to the Cq value (i.e., the cycle number at which fluorescence has increased above background levels) from the qPCR (rounded up). Library amplification was followed by SPRI (Beckman Coulter) size selection to exclude fragments larger than 1,200 bp. DNA concentration was measured with a Qubit fluo-rometer (Life Technologies). Library amplification was performed using custom Nextera primers (Buenrostro et al., 2013). The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 3000/4000 platform and the 50-bp single-end configuration.