GSM5493339: CLAP-seq_INPUT_dRRM_B [CLAP-seq]; Mus musculus musculus x M. m. castaneus; OTHER
Cells were lysed in iCLIP lysis buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% Sodium Deoxycholate) supplemented with 1X Protease Inhibitor Cocktail (Promega), 200 U of Murine RNase Inhibitor (New England Biolabs), 20 U Turbo DNase (Ambion), and 1X Manganese/Calcium Mix (0.5mM CaCl2, 2.5 mM MnCl2). RNA was dephosphorylated (Fast AP) and cyclic phosphates removed (T4 PNK). The RNA was then ligated to an RNA adapter containing a RT primer binding site. The ligated RNA was reverse transcribed (RT) into cDNA, the RNA was degraded using NaOH, and a second adapter was ligated to the single stranded cDNA. The DNA was amplified and Illumina sequencing adaptors were added by PCR using primers that are complementary to the 3' and 5' adapters.