GSM5905473: ChIP_WT_H3K14ac; Mus musculus; ChIP-Seq
For ChIP, ESCs were fixed in 1% formaldehyde, followed by quenching and cell lysis before chromatin sonication using a E220 focused-ultrasonicator (Covaris). Sheared chromatin was then subjected to immunoprecipitation, and immunoprecipitated DNA was then washed, eluted, reverse cross-linked, and purified prior to submission for library preparation. Spike-in normalized ChIP samples were mixed at a 2:1 cell count ratio (mammalian: fly) with Drosophila melanogaster S2 cells crosslinked the same way, prior to sonication. RNA was extracted using RNeasy mini kit (Qiagen) following manufacturer's instructions. All samples were lysed in 350ul of QIAGEN Buffer RLT prior to extraction. ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and IP samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250- 450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit. RNA-seq libraries were prepared using the Illumina TruSeq Stranded Total RNA Preparation Kit. Ribosomal RNA was depleted using QIAseq FastSelect –rRNA HMR Kit per manufacturer's instructions. Input RNA quality was validated using the Agilent RNA 6000 Nano Kit. 200ng-1 µg of total RNA was used as starting material. Libraries were validated using the Agilent DNA 1000 Kit.