GSM6784625: Chd4_shRNA_Rep2; Mus musculus; RNA-Seq
To quantify nascent RNA synthesis affected by desired treatment, i.e., BRG1 inhibition or Chd4 shRNA KD, mouse ES cells were incubated with 500 µM 4-thiouridine for 5 min at 37°C and 5% CO2 and lysed in TRIzol which was then spiked in with external reference RNA (2.4 ng synthetic ERCC RNA mixture for per million mESC). Total RNA was extracted using phenol-chloroform method and precede to biotinylating by incubation with HPDP-Biotin for 2 hours. Nascent RNA was isolated from unlabeled RNA by streptavidin beads and eluted in 100 mM DTT followed by purification with MACHEREY-NAGEL NucleoSpin RNA kit according to the manufactory's instruction. Nascent RNA libraries were prepared using NEBNEXT Ultra II directional RNA preparation kit (E7760S) according to manufactory's instruction, briefly, nascent RNA with different length were fragmentated into an average of 300 nt and reverse transcribed into stranded DNA using random primers, which was followed by 3-prime end indexing during PCR amplification.