GSM7876835: nascentRNA_FX4; Mus musculus; RNA-Seq
Skeletal muscle for ChIP-Seq was dissected and finely minced. ChIP-seq samples were subjected to cross linking in 1% formaldehyde for 30 minutes and quenched with glycine. Tissue was dounced to release nuclei. Nuclei were sonicated to shear the chromatin and subjected to immunoprecipitation. Precipitates were washed and DNA was purified by column (Qiagen Min-Elute). Tissues for RNA-Seq were homogenized in Trizol using a MoBio PowerLyzer 24. Lysates were purified via Qiagen RNAeasy purification column. RiboSeq samples pulverized in liquid nitrogen then lysed in polysome lysis buffer supplemnted with 100ug/ml cyclohexamide. Lysate were dounced, then cleared by centrifugation. Cleared lysates were next treated with 3000U of T1RNase and ribosomes immunoprecipitated with HA tag magnetic beads. RNA was extracted from the immunoprecipitated ribosomes and subjected to library prep. ChIP-seq libraries were prepared with the KAPA Hyper Prep Kit (Kapa Biosciences). RNA-seq libraries were prepared with the Illumina Tru-seq stranded mRNA kit. Ribo-seq libraries were prepared with the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs).