GSM5068656: Arid1a_wt_rep3; Mus musculus; ATAC-seq
The pancreas of 8-week-old p48-Cre and Arid1afl/fl;p48-Cre mice were harvested in ice-cold HBSS, dissociated by surgical scissors in a 10 cm cell culture dish filled with 5 ml cold HBSS and centrifuged at low speed (300 rpm, 5 min) to remove blood vessels and adipose tissue. The remaining explant was digested in 5 ml solution 2 at 37 ºC for 20 min, pipetted every 2 min using a 10 ml pipette. The tissue-containing tube was washed with 10 ml solution 1 and centrifuged (300 rpm, 5 min) before adding a second time of solution 2 and cultivated at 37 ºC for 10 min. Next, tissue suspension was transferred into a new 50 ml tube by filtering through a 100 µm cell strainer and the tissue fragments were ground using a syringe plunger. The remaining cells were collected by washing the mesh with two times of 10 ml solution 1 and pelleted. Recovering was achieved by easing the acinar cells in 5 ml solution 3 containing 10% FCS for 1 hour at 37 ºC and subjected to ATAC-seq library preparation. ATAC-seq library preparation was conducted using Nextera DNA Library Prep (Illumina) as previously described (Buenrostro et al., 2013). 10,000 cells were used for the assay. Crude nuclei preparation was achieved by incubating the cells in ATAC lysis buffer. The pelleted nuclei were incubated with transposition reaction buffer containing Tn5 transposases and incubated at 37 ºC for 30 min, followed by DNA purification using Qiagen MinElute PCR purification Kit. Further, transposed DNA was barcoded and amplified through PCR. After quality control by Agilent Bioanalyzer 2100 and size-selection, samples were prepared as 2 nmol/L and sequenced on a HiSeq 2500