GSM5571799: F1648 EtOH_Hdac2 rep1; Mus musculus; ChIP-Seq
10 million cross-linked cells per sample were lysed in 5 Aliquots in Lysis Buffer I on ice for 10 min, spun down and supernatant was removed. Next, pellet was resuspended Lysis Buffer II on ice for 10 min, spun down and supernatant was removed. Then, the pellet was resupended in 300 µl Shearing Buffer and incubated on ice for 10 min and sonicated in a Bioruptor Plus TPX microtubes using a Bioruptor device to shear the chromatin to an average size of 100–500 base pairs long. After successful size check, sonicated chromatin was precleared with blocked Dynabeads 5% of precleared sample was used for Input. Then of sonicated chromatin was incubated overnight at on a rotating wheel with Hdac2 antibody. The next day, blocked beads were added to antibody-bound chromation and incubated at 4°C for 2 hours. Next, beads were washed twice with 1ml Low Salt Wash Buffer. Low-binding Eppendorf tubes were changed after first wash. Then, beads were washed with High Salt Wash Buffer. Next, beads were washed with LiCl Wash Buffer. Then, 1x TE-buffer was added and beads were placed on a magnet and supernatant was aspirated completely. For crosslink reversal of immunoprecipitated DNA beads were resuspended in ChIP Elution Buffer. Input sampes were diluted with Chip Elution Buffer. Next RNAse A was added and Samples and Input Chromatin were incubated at 37°C for 30 min. Afterwards, Proteinase was added and beads or Input Chromatin were incubated at 55°C for 1h and then incubated at 65°C for 8h for decrosslinking. Afterwards beads were separated from supernatant using a magnet and supernatant was collected in a new tube. Elution buffer was added to the beads and incubated for one minute and then elution buffer was combined with supernatant. Proteinase K was added to eluates and incubated at 55°C for 1 h. For DNA Purification, Ampure Beads were added to the decrosslinked Chromatin samples. Entire Volume was then mixed for at least 10 times and beads and DNA was incubated at room temperature for 10 min. Afterwards tubes were placed on a magnet and supernatant was removed. Next, Beads were washed with 80% Ethanol twice. Beads were then dried for 5 minutes before adding Elution buffer. Beads were incubated for 5 minutes and Eluate was collected in a new tube. Eluate concentrations were determined using Qubid HS DNA Assay. Purified DNA was used as input for library preparation with NEBNext Ultra II DNA Library Prep Kit for Illumina and processed according to the manufacturer´s instructions. Libraries were quality controlled by Qubit and Agilent DNA Bioanalyzer analysis. The library was sequenced on a NextSeq 500 (Illumina) with 76 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2.