GSM8832232: iPSC-CM_IndA_DNR_3h_H3K27ac_CT; Homo sapiens; OTHER
Samples were fast-thawed and centrifuged at 4 °C for four min at 1000 x g. The supernatant was poured off and the cell pellet retained. The pellets were resuspended and washed twice in 1 mL of Wash Buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Protease inhibitor cocktail; Roche 11873580001). Nuclei were extracted by incubating cells for 10 min on ice in 200 µL/sample of cold Nuclei Extraction buffer (NE buffer: 20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol, 0.5 mM Spermidine, 1x Protease Inhibitor cocktail). Following incubation, nuclei were centrifuged at 4 °C for four min at 1300 x g and drained by inverting onto a paper towel. The cells were resuspended in PBS and fixed with 0.1% formaldehyde solution for two min at room temperature, with the termination of cross-linking by adding 2.5 M glycine to twice the molar concentration of formaldehyde. After fixation, the nuclei were centrifuged at 4 °C for four min at 1300 x g, and the liquid was poured off through inversion over a paper towel. The pellet was resuspended in 1.5 mL of Wash Buffer. Concanavalin A coated magnetic beads (Bangs Laboratories Inc, BP531; Lot 16963) were prepared as previously described (Skene, Elife, 2017) and 10 µL of activated beads were added to each sample and incubated for 15 min at room temperature on an end-over-end rotator. Beads with sample were collected via magnet and unbound supernatant was discarded. The sample was resuspended in 50 µL of ice-cold Antibody Buffer (Wash Buffer with 2 mM EDTA and 0.1% BSA) with appropriate antibody dilution (H3K27ac, Active Motif, 39034; Lots 31521015, manufacturer recommendation of 1:50). Samples were incubated at 4 °C overnight on a tilt table. After incubation, the primary antibody was removed by magnetic pelleting to clear the supernatant and discarding the non-bound supernatant. Each pellet was resuspended in 100 µL of secondary antibody, Guinea Pig anti-Rabbit IgG (Heavy & Light Chain) Preabsorbed Antibodies (Antibodies-Online ABIN101961; Lot NE-200-032309), diluted 1:100 in Wash Buffer. The samples were incubated for one hr at room temperature on a tilt table. The secondary antibody was removed using a magnet stand to clear the supernatant. The bound samples were washed twice with 1 mL of Wash Buffer for five min each wash. After washing, 50 µL of a solution of pAG-Tn5 (CUTANA™ pAG-Tn5, Epicypher 15-1017; Lot 23243004-C1) diluted 1:20 in 300 Buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1× Protease inhibitor cocktail) was added to each sample and samples were incubated on a tilt table for one hr at room temperature. Samples were collected using a magnet stand and washed twice with 1 mL of 300 Buffer. Next, samples were resuspended in 300 µL Tagmentation Buffer (10 mM MgCl2 in 300 Buffer) and incubated in a water bath at 37 °C for one hr. To stop tagmentation, 10 µL of 0.5 M EDTA, 3 µL of 10% SDS, and 2.5 µL of 20 mg/mL Proteinase K were added to each 300 µL of sample, vortexed and incubated in a water bath at 55 °C for one hr. DNA was extracted using PCI-Chloroform separation, extracting the aqueous layer, placing it in a new tube, discarding the rest. The samples were chilled on ice and centrifuged at 4 °C for 10 min at 16,000 x g. The liquid was decanted, and pellets were resuspended with 100% ethanol, followed by centrifugation at 4 °C for one min at 16,000 x g. Supernatant was decanted, and pellets were allowed to air dry before dissolving in 30 µL TE buffer (1 mM Tris-HCl pH 8, 0.1 mM EDTA). To amplify and index libraries, 21 µL of DNA was combined with 2 µL of a 10 µM uniquely barcoded i5 and 2 µL of a uniquely barcoded i7 primer (Buenrostro, Nature, 2015), using a different barcode combination for each sample. A volume of 25 µL NEBNext HiFi 2× PCR Master mix was added and mixed. The sample was placed in a thermocycler with a heated lid using the following cycling conditions: 72 °C for five min (gap filling); 98 °C for 30 s; 14 cycles of 98 °C for 10 s and 63 °C for 10 s; final extension at 72 °C for one min and hold at 8 °C. Post-PCR clean-up was performed by adding 1.3× volume of AMpureXP beads (Beckman Counter), and libraries were incubated with beads for 10 min at room temperature, washed twice gently in 80% ethanol, and eluted in 25 µL 10 mM Tris pH 8.0. Libraries additionally underwent a double-sided clean-up to eliminate the large and small fragments, using AMpureXP beads at a 0.55 x and then 1.8 x ratio, respectively.