title: GSM7596700: NMR, Heart, Igf1r, NMR35, vl0451; Heterocephalus glaber; OTHER
library_construction_protocol: Tissues were prepared by direct perfusion of the liver with PBS, followed by dicing the whole organs (liver and heart) in small pieces around 1cm3. Blood clots within the heart ventricles were removed. Cross-linking of the diced tissue was performed in 1% formaldehyde solution for 20 min, addition of 250 mM glycine and incubation for a further 10 min to neutralize the formaldehyde. After homogenization of cross-linked tissues in a dounce tissue grinder, samples were washed twice with PBS and lysed according to published protocols (SCHMIDT et al. 2009 for ChIP and Krijger et al. 2020 for 4C) to solubilize DNA-protein complexes. For ChIP, chromatin was fragmented to 300 bp average size by sonication on a Misonix sonicator 3000 with a 418 tip (1/16 inch diameter). Chromatin from 50-200 mg of dounced tissue was used for each ChIP experiment using antibodies against H3K4me3 (millipore 05-1339), H3K27ac (abcam ab4729), H3K4me1 (abcam ab8895), TBX5 (Insight bio sc-515536), RXRA (sc-553), FOXA1 (ab70382), HNF4A (ARP31946), CEBPA (sc-9314) and HNF6 (sc-13050). For 4C, locus-specific 4C templates were made using two rounds of enzyme digestion (primary and secondary) and ligation. Purified 4C templates were amplified by inverse PCR with bait-specific primers. Illumina sequencing libraries were prepared from ChIP-enriched or 4C-enriched DNA using ThruPLEX DNA-seq library preparation kit (Takara Bio) or NEBNext Ultra II DNA Library Prep Kit for Illumina with up to 10ng of input DNA and 8-15 PCR cycles. After PCR, libraries were pooled in equimolar concentrations and sequenced on Illumina HiSeq 4000, NextSeq 500 or NovaSeq 6000 instruments. ChIP-Seq, 4C-Seq