title: GSM3756550: A-Fos (+) CMs ATAC-Seq rep2; Danio rerio; ATAC-seq
library_construction_protocol: Adult ventricles were dissected in 1x HBSS (Gibco) and minced into 1-2 mm pieces using microdissecting spring scissors.  Minced ventricle pieces from 6 adult hearts were washed twice in 1x HBSS, HBSS was removed and ventricle pieces were dissociated by enzymatic digestion in 90 μL Cardiomyocyte Isolation Enzyme #1 (Thermo Fisher Scientific) and 4.5 μL Cardiomyocyte Isolation Enzyme #2 (Thermo Fisher Scientific).  Enzymatic digestion was performed at 37°C for 30 minutes, followed by two washes in HBSS and resuspension in tissue culture medium (DMEM/F-12 1:1, 10% FBS, 1x Penicillin-Streptomycin).  Uninjured Tg(myl7:nucDsRed)+ or Tg(gata4:EGFP)+ cardiomyocytes at 4 dpci were isolated by FACS sorting using a FACS Aria III.  CM:A-Fos (+) and CM:A-Fos (-) cardiomyocytes at 4 dpci and uninjured CM:junbaOE and CM:junbbOE at 7 days post induction of overexpression were isolated by density gradient centrifugation according to (Golden et al., 2012). Cardiomyocytes were harvested by centrifugation at 500 g for 5 minutes at 4°C.  Cells were washed once with 50 μL of cold PBS and spun down at 500 g for 5 minutes at 4°C.  Cell pellets were resuspended in 50 μL of cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) and spun down immediately at 500 g for 10 minutes at 4°C.  For 50000 cells, nuclei were gently resuspended in 25 μL 2x TD Buffer (Illumina #FC-121-1030), 2.5 μL Tn5 Transposase (Illumina #FC-121-1030) and 22.5 μL nuclease-free H2O and incubated at 37°C for 30 minutes.  Immediately following transposition, transposed DNA was purified using a PCR purification MinElute kit (Qiagen) and eluted in 10 μL nuclease-free H2O.  For 5000 cells, nuclei were resuspended 5 μL 2x TD Buffer, 0.35 μL Tn5 Transposase, and 4.65 μL nuclease-free H2O.  Transposition reactions were incubated at 37°C for 30 minutes, followed by the addition of 1.2 μL 500 mM EDTA and incubation at 50°C for 30 minutes.  1.2 μL of 500 mM MgCl2 was added to each reaction and no purification was performed prior to PCR amplification.  Transposed DNA fragments were amplified using NEBNext High-Fidelity 2x PCR Master Mix (NEB #M0541) and 1.25 μM Nextera bar-coded primers with the following cycling conditions: 1x: 72°C, 5 mins/1x: 98°C, 30 seconds/5x: 98°C, 10 seconds, 63°C, 10 seconds, 72°C, 1 minute.  qPCR was performed to determine the optimal number of additional cycles according to (Buenrostro et al., 2015) and additional cycles were performed as above.  Amplified library DNA was purified using a PCR purification MinElute kit (Qiagen) according to manufacturer's instructions and an additional clean-up step was performed to remove excess primers with 1:1 (vol/vol) AMPure XP beads (Beckman Coulter) according to manufacturer's instructions.