title: GSM9380177: Pooled_Input; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP-sequencing was performed by Active motif (Carlsbad, CA, USA) using young WT and NADS KO gastrocnemius muscles. Muscles were mechanically homogenized and after fixation with 1% formaldehyde quenched with glycine. Lysis buffer was used to isolate the chromatin. Lysates were sonicated to sheared DNA with an average length of 300-500 bp with Active Motif's PIXUL® Multi-Sample Sonicator. Isolated chromatin was treated with RNase, proteinase K, and heat for de-crosslinking to yield Genomic DNA. Quantification was performed by Qubit Flex Fluorometer (Thermo Fisher Scientific, Inc., MA, USA). Chromatin aliquot of 30 µg, spiked-in with 35 µl of Drosophila chromatin was precleared to remove nonspecific ligands and proteins via protein G coupled agarose beads (Invitrogen, MA, USA). Genomic DNA regions of interest were isolated using 4 µl of antibody against H3K27me3 (Active Motif). Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 °C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. ChIP sequencing libraries were quantified and sequenced on Illumina's NovaSeq 6000.