GSM2064244: PA1 4sU 16 hr ribo-RNA replicate 1; Homo sapiens; RNA-Seq
100-140 μg total RNA was used for biotinylation and purification of 4sU labeled nascent RNA. 4sU labeled RNA was biotinylated in biotinylation buffer (10 mM Tris pH 7.4, 1 mM EDTA) with 0.2 mg/ml EZ-link biotin-HPDP (Pierce, 21341), which was dissolved in dimethylformamide (DMF, Sigma, D4551) at a concentration of 1 mg/ml, and incubated at room temperature for 1.5 h with rotation. Unbound biotin-HPDP was removed by equal volume chloroform extraction twice, and RNA was precipitated at 1,3000 rpm for 15min at 4℃ with 1:10 volume of 5 M NaCl and an equal volume of isopropanol. The RNA pellet was washed twice with 75% ethanol and resuspended in 150 μl DEPC•H2O. RNA quality was checked by electrophoretical analysis to exclude RNA degradation. 4sU labeled and unlabeled RNA was separated by using Streptavidin-coated magnetic beads (Invitrogen). Biotinylated RNA was incubated with 150 μl Streptavidin beads at room temperature for 20 min. Beads were washed four times with 0.9 ml of 65 ℃ washing buffer (100 mM Tris pH 7.4, 10 mM EDTA, 1 M NaCl, 0.1% Tween 20), followed by four times with 0.9 ml of room temperature washing buffer. Nascent RNA was eluted with 100 μl 0.1 M dithiotheitol (DTT) twice, and RNA was precipitated with 40 μl of 4 M LiCl, 2 μl glycogen and 600 μl ice-cold ethanol. Before preparation of nascent RNA-seq library, ribosomal RNA was depleted from 4sU labeled RNAs with RiboMinusTM Human/Mouse Transcriptome Isolation Kit (Invitrogen, K1550-01) according to the manufacturer’s protocol. 4sUDRB and ribo-RNA-seq libraries were prepared with Illumina TruSeq Total RNA LT Sample Prep Kit without oligo(dT) enrichment. Deep sequencing was performed on Illumina Hiseq 2500 at CAS-MPG Partner Institute for Computational Biology Omics Core, Shanghai, China.
GEO Accession
GSM2064244