title: GSM3684424: CLIP-seq_HSV-1-2; Homo sapiens; OTHER
library_construction_protocol: (1) For 4SU-seq: 4sU was added to 5 ml medium at 500 μM for each 10 cm dish and incubated for 1 hr before cells were harvested. For HSV1-infected cells, 4sU was added at 7 hr post infection. Discard medium and add 3 ml Trizol (Invitrogen) in dark. Total RNA were isolated from the cells as per instruction. 700 μl thiol-specific biotinylation reaction contained 70 μg total RNA, 70 μl 10x Biotinylation Buffer (100 mM Tris-Cl pH 7.5 and 10 mM EDTA), 140 μl biotin-HPDP (in 1 mg/ml DMF), 70 ul DMF. Incubate the reaction at room temperature for 1.5 hr with rotation. Add an equal volume of chloroform. Mix vigorously. Centrifuge at 14,000 rpm for 10 min at 4 °C and transfer aqueous phase into a new tube. Repeat chloroform extract once. Add 1/10 the volume of 5 M NaCl and an equal volume of isopropanol to precipitate RNA. Centrifuge at 14,000 × g for 20 min at 4 °C. Discard supernatant and wash once with 75% ethanol. Re-suspend biotinylated RNA in 100 μl H2O. Wash 50 μl streptavidin-magnetic beads (Invitrogen) with solution A (0.1 M NaOH and 0.05 M NaCl) for 2 x 2 min followed by solution B (0.1 M NaCl) for 2 x 2 min. Keep beads in 100 μl 2 x B&W buffer (10 mM Tris-Cl pH 7.5, 1 mM EDTA and 2 M NaCl). Heat 100 μl biotinylated RNA samples to 65 °C for 10 min to denature and immediately place on ice. Add 100 μl pre-washed streptavidin-magnetic beads  to roate for 1hr. Wash beads with 800 μl 1 x B&W buffer (5 mM Tris-Cl pH 7.5, 0.5 mM EDTA and 1 M NaCl) for 3 x 5 min and incubate beads-4SU-RNA with 100 μl 0.1 M DTT for 2 x 5 min at room temperature. Add 1/10 the volume of 5 M NaCl and an equal volume of isopropanol and 1ul glycoblue to precipitate RNA. Centrifuge at 14,000 × g for 20 min at 4 °C. Discard supernatant and wash once with 75% ethanol. Re-suspend newly transcribed RNA in 20 μl H2O for libraries preparation. (2) For CLIP-seq: The approach is modified from eCLIP. Briefly, Infected and transfected Hela cells were exposed to 254 nM UV with 400 mJ/cm2. Cells were scrapped and re-suspended in lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate and Protease Inhibitor). After RNase I digestion, 80 μl of protein G Dynabeads beads and 5 μg ICP27 antibody per IP were used for immunoprecipitation overnight. After washing with High salt buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS and 0.5% sodium deoxycholate) and wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2 and 0.2% Tween-20), phosphates at 5' and 3' end of RNA were removed by CIP for 15 min at 37oC . 3' RNA linker was added by T4 RNA ligase overnight and [γ-32P]ATP was used to label 5' end of RNA by T4 PNK. ICP27/RNA complex was resolved in SDS-PAGE and transfered to NC membrane. Proper regions of membrane were cut out to extract RNA in PK/Urea buffer. Extracted RNA was added with 5' RNA linker by T4 RNA ligase and reverse-transcribed with Superscript III. Libraries for CLIP-seq were got after PCR using illumina universal primer and index primers. (1) For 4SU-seq: Libraries were directly fragmented and prepared according to Illumina's instructions; (2) For CLIP-seq: RNA linkers for CLIP-seq are designed according to illumina universal primers and index primers, and the final libraries were got by PCR.
identifiers.namespace: GEO
identifiers.id: GSM3684424