title: GSM7697980: Input-PN1; Mus musculus; ChIP-Seq
library_construction_protocol: For each sample, ventricles from 2 mouse hearts were used as biological duplicates for the ChIP-Seq experiments. Tissue samples were ground to powder and crosslinked with 2% formaldehyde in PBS for 30 min followed by quenching with 0.125 M glycine for 10 min at room temperature. Crosslinked samples were then washed with cold 1X PBS and further homogenized with a Dounce tissue grinder. Samples were collected by a brief spin and treated with 10 mM Tris-HCl (pH 8.0), 10mM NaCl and 0.2% NP-40 for 30 min to collect nuclei. After nuclear extraction, chromatin was sheared on a Bioruptor Pico (Diagenode) for 20 cycles (30 sec on/ 30 sec off for each cycle) at 4°C in sonication buffer (0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl, 1 mM EDTA, 0.1% sodium deoxycholate, 0.25% sarkosyl, 1 mM DTT, 1x cOmplete Protease Inhibitor Cocktail (Roche), and 200 µM PMSF, pH 8.0). 1% of the sonicated chromatin from each sample was taken out as “input” samples at this step. A sonication buffer was added to the remaining sonicated chromatin to make the final volume 1 mL. NaCl was then added to a final concentration of 300 mM for each sample. Meis1/2 (Santa Cruz, sc-10599X; 1:200) or Hoxb13 (GeneTex, GTX129245; 1:100) was added to each sample and incubated at 4°C overnight with gentle rotation. The next day, 30 µL of pre-washed Dynabeads Protein G (Invitrogen, 10004D) was added to each sample for a two-hour incubation. After that, the beads were washed twice with 1 mL RIPA 0 buffer (0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl, 1 mM EDTA, 0.1% sodium deoxycholate, pH 8.0), twice with 1 mL RIPA 0.3 buffer (0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl, 1 mM EDTA, 0.1% sodium deoxycholate, 300 mM NaCl, pH 8.0), twice with 1 mL LiCl wash buffer (250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0), and finally twice with 1 mL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). For each ChIP sample or input sample, 100 uL of SDS elution buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0) was added and incubation was done at 65°C overnight on a ThermoMixer (Eppendorf) at 1000 rpm. The next day, supernatant was collected and further treated with 0.5 µg RNaseA (Sigma, 11119915001) for 30 min at 37°C, followed by 20 µg Proteinase K (NEB, P8107S) treatment at 37°C for 2h. DNA was recovered using MinElute PCR Purification Kit (QIAGEN, 28004) according to manufacturer's protocol. ChIP–seq libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) according to manufacturer's protocol. Final ChIP–seq libraries were pooled and sequenced on an Illumina Nextseq 500 system using the 75-bp high output sequencing kit for single-end sequencing