title: GSM9013556: Rdh10 KO input; Mus musculus; ChIP-Seq
library_construction_protocol: For ChIP-seq we used trunk tissue from E10 wild-type or Rdh10-/- embryos dissected in modified PBS, i.e. phosphate-buffered saline containing 1X complete protease inhibitors (concentration recommended by use of soluble EDTA-free tablets sold by Roche #11873580001) and 10 mM  sodium butyrate as a histone deacetylase inhibitor (Sigma # B5887). Samples were processed similar to previous methods (Berenguer et al., PLOS Biology 2020). Dissected face tissues were briefly centrifuged in 1.5 ml tubes and excess PBS dissection buffer was removed. For cross-linking of chromatin DNA and proteins, 500 ml 1% formaldehyde was added, the trunk samples were minced by pipetting up and down with a 200 ml pipette tip and then incubated at room temperature for 15 min. To stop the cross-linking reaction, 55 ml of 1.25 M glycine was added and samples were rocked at room temperature for 5 min. Samples were centrifuged at 5000 rpm for 5 min and the supernatant was carefully removed and discarded. A wash was performed in which 1000 ml of ice-cold modified PBS was added and mixed by vortex followed by centrifugation at 5000 rpm for 5 min and careful removal of supernatant that was discarded. This wash was repeated. Cross-linked trunk samples were stored at -80C until enough were collected to proceed, i.e. 100 wild-type trunks and 100 Aldh1a2-/- trunks to perform ChIP-seq with two antibodies in duplicate. Chromatin was fragmented by sonication. Cross-linked trunk samples were pooled, briefly centrifuged, and excess PBS removed. 490 ml lysis buffer (modified PBS containing 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0) was added, mixed by vortexing, then samples were incubated on ice for 10 min. Samples were divided into four sonication microtubes (Covaris AFA Fiber Pre-Slit Snap-Cap 6x16 mm, #520045) with 120 ml per tube. Sonication was performed with a Covaris Sonicator with the following settings - Duty: 5%, Cycle: 200, Intensity: 4, #Cycles: 10, 60 sec each for a total sonication time of 14 min. The contents of the four tubes were re-combined by transfer to a single 1.5 ml microtube which was then centrifuged for 10 min at 13,000 rpm and the supernatants transferred to a fresh 1.5 ml microtube. These conditions were found to shear trunk DNA to an average size of 300 bp using a 5 ml sample for Bioanalyzer analysis. At this point 20 ml was removed for each sample (wild-type trunks and Aldh1a2-/- trunks) and stored at -20C to serve as input DNA for ChIP-seq. Each sample was divided into four 100 ml aliquots to perform immunoprecipitation with ChIP-grade antibodies for H3K27ac (Active Motif, Cat#39133) in triplicate. Immunoprecipitation was performed using the Pierce Magnetic ChIP Kit (Thermo Scientific, #26157) following the manufacturer's instructions. The immunoprecipitated samples and input samples were subjected to reversal of cross-linking by adding water to 500 ml and 20 ml 5 M NaCl, vortexing and incubation at 65C for 4 hr; then addition of 2.6 ml RNase (10 mg/ml), vortexing and incubation at 37C for 30 min; then addition of 10 ml 0.5 M EDTA, 20 ml 1 M Tris-HCl, pH 8.0, 2 ml proteinase K (10 mg/ml), vortexing and incubation at 45C for 1 hr. DNA was extracted using ChIP DNA Clean & Concentrator (Zymo, # D5201 & D5205), After elution from the column in 50 ml of elution buffer, the DNA concentration was determine using 2 ml samples for Bioanalyzer analysis. The two input samples ranged from 16-20 ng/ml and the eight immunoprecipitated samples ranged from 0.1-0.2ng/ml (5-10 ng per 100 trunks). For ChIP-seq, 2 ng was used per sample to prepare libraries for DNA sequencing.