GSM8641794: hiPSCNeuronsJUNWT_vinc_ChIP_FLAG_rep2; Homo sapiens; ChIP-Seq
Cell pellets were thawed on ice for 10 minutes. Cells were lysed and sonicated using the truChIP Chromatin Shearing Kit (Covaris 520154) according to the manufacturer's instructions. Chromatin was sonicated in Covaris milliTUBE 1 ml AFA tubes under the following conditions: 30 minutes total, PIP 75, duty factor 15%, CPB 1000, using a Covaris ME220 sonicator. Sonicated samples were centrifuged at 20,000 g for 15 minutes at 4°C, and the supernatant was collected. Chromatin concentration was determined using the Qubit high sensitivity DNA kit. A 20 µg aliquot of chromatin was used per 1 ml ChIP reaction, adjusted with RIPA buffer. For immunoprecipitation, 30 µl of Protein A/G beads conjugated with 2 µg of antibody (pre-incubated with beads resuspended in 500 µl RIPA for 3 hours) was added, and the mixture was incubated overnight at 4°C on a rotating platform. The beads were then washed once with low-salt wash buffer, twice with high-salt wash buffer, twice with lithium chloride wash buffer, and twice with 1X TE buffer. Chromatin was eluted in 200 µl of freshly prepared direct elution buffer supplemented with 2 µl RNase A (10 mg/ml) and incubated on a thermomixer at 65°C and 800 rpm overnight. The following day, beads were removed, and the supernatant was treated with 3 µl Proteinase K (20 mg/ml) at 55°C and 1200 rpm for 2 hours to reverse crosslinks. DNA was purified using the Zymo DNA Clean and Concentrator kit (Zymo, D4033) and eluted in 20 µl deionized water. ChIP-seq libraries were prepared following the SMARTer ThruPlex TAKARA Library Prep kit. 10 µl of purified DNA was used per sample. The DNA was prepared with Template Preparation D Buffer (2 µl) and Template Preparation D Enzyme (1 µl) and subjected to end repair and A-tailing in a thermocycler. Library synthesis was performed by adding Library Synthesis D Buffer (1 µl) and Library Synthesis D Enzyme (1 µl) followed by library amplification using Library Amplification D Buffer (25 µl), Library Amplification D Enzyme (1 µl), Nuclease-free water (4 µl), and an Illumina-compatible indexed primer (5 µl). Amplification consisted of an initial extension at 72°C for 3 minutes, denaturation at 98°C for 2 minutes, followed by 4 cycles of 98°C for 20 seconds, 67°C for 20 seconds, and 72°C for 10 seconds for index addition, and 12 cycles of 98°C for 20 seconds and 72°C for 10 seconds for amplification. Libraries were cleaned using a SPRIselect bead double size selection and sequenced on a NovaSeq platform using PE50.