GSM8774056: mES H3K9me2 b1; Mus musculus; OTHER
Whole hearts and right forebrain lobes were collected and pooled by tissue type from the remaining embryos. Each tissue or cell type consisted of 3 biological replicates (tissues from 3 separate pooled litters or 3 separate culture vessels). Mouse embryonic stem cells (mESCs) or freshly isolated tissues were dissociated into single cell suspensions (via TrypLE (Gibco, cat# 12605-010) for mESCs or papain (Worthington, cat# LK003150) for tissues). CUT&RUN was then performed per the instructions included in the CUTANA ChIC/CUT&RUN Kit (EpiCypher, cat# 14-1048, v1.0-v3.0). In brief, cells were washed and then allowed to bind to activated ConA beads. Samples were then incubated overnight at 4C on a nutator with the appropriate antibody. The following day samples were washed again and then incubated with pAG-MNase for 10 minutes at room temperature. Samples were washed again, and targeted chromatin digestion was initiated with 100mM calcium chloride and allowed to proceed for 2 hours at 4C on a nutator. The reactions were then stopped, and DNA fragments were collected via incubation in a thermocycler set to 37C for 10 minutes. Finally, DNA fragments were purified via the NucleoSpin Gel and PCR Clean-up kit (Takara Bio USA, Inc, cat# 740609). DNA libraries for Next-Generation sequencing were prepared utilizing the KAPA HyperPrep Kit (Kapa Biosystems, cat# KK8502 07962347001) and NEBNext Multiplex Oligos for Illumina (New England Biolabs, cat# E6440S). To begin, samples were end repaired & A-tailed via incubation in a thermocycler for 30 minutes at 20C followed by a 58C incubation for 60 minutes. Next adapters were ligated per kit instructions with a 15-minute incubation at 20C with ligation buffer & DNA ligase, followed by a 37C incubation for 15 minutes with NEB USER enzyme for an additional 15 minutes. Post-ligation cleanup was performed using 1.1x AngenCourt AMPure XP Beads (Beckman Coulter, cat# A63880). Next, the libraries were amplified per kit instructions with the following thermocycler conditions: 1 cycle of initial denaturation at 98C for 45 seconds, 14 cycles of denaturation at 98C for 15 seconds and annealing and extension at 60C for 10 seconds, and 1 final extension cycle at 72C for 1 minute. Amplification was followed by an additional cleanup step with the 1.1x AngenCourt AMPure XP Beads and library quantification using the NEBNext Library Quantification Kit (cat# E7630) as per kit instructions. Library quality and fragment size was then determined via Agilent Technologies TapeStation using the High Sensitivity D1000 ScreenTape (cat# 5067-5584) and reagents (cat# 5067-5585). Libraries were then pooled and denatured via the standard Illumina protocol. Finally, samples were paired-end sequenced on an Illumina NextSeq 500 with a high-throughput flow cell (cat# 20022408).