GSM5012386: Nfia_rep1_RNA-seq; Mus musculus; RNA-Seq
For ChIP-seq, cells were fixed with 1% formaldehyde-containing medium and suspended in lysis buffer. The lysates were sonicated with a sonicator. After centrifugation, the supernatant was collected. Dynabeads M-280 sheep anti-mouse IgG (Thermo Fisher, Cat# DB11202), with magnetic property, were pre-incubated with 5 μg of anti-Flag antibody (Sigma-Aldrich, Cat# F3165) and then incubated with sonicated chromatin with rotation at 4℃ overnight. The immunoprecipitated samples were washed and then eluted in elution buffer at 65℃ overnight. Samples were treated with RNase A at 37℃ for 1 h, followed by proteinase K at 55℃ 2 h. DNA was purified according to conventional PCI method. For RNA-seq, total RNA was extracted using Sepasol®-RNA I Super G (nacalai tesque, Cat# 09379-55), and then mRNA was further isolataed with NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Cat#E7490) according to their standard procedure. For ChIP-seq, libraries were made according to instructions in the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Cat# E7645S). For RNA-seq, purified mRNAs were supplied for library construction using the NEBNext Ultra Directional RNA library Prep Kit for illumine (New England and Biolabs, Cat# E7420) according to manufacturer's protocols.