title: GSM2416840: HeLa scramble ribo–/poly(A)– RNA replicate 2; Homo sapiens; RNA-Seq
library_construction_protocol: For total RNA, 10μg of total RNAs isolated from HeLa cells were separated into polyadenylated and nonpolyadenylated fractionations by oligo(dT) magnetic beads (Invitrogen). The polyadenylated RNA population was purified by oligo(dT) magnetic beads and followed by DNase I treatment at 37 °C for 30 mins (Ambion, DNA-freeTM Kit). Ribosomal RNAs were further depleted by using RiboMinus kit (Invitrogen) (Yang et al., 2011) to get the poly (A)-/ribo- RNA population. Then poly (A)-/ribo- RNAs were subjected to Trizol extraction. For nascent RNA, 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. For iCLIP-seq, The UV-crosslinked cells were resuspended in lysis buffer and sonicated for 20s x 6 times on 25% duty. by immunoprecipitaiton with 8 µg anti-Flag (ShangHai Genomics) antibody at 4°C for 2 hours. Beads with protein-RNA complexes with 5 µg yeast tRNAs were treated with 5 Kunz U/ml of micrococcal nuclease for 5 min at 37°C, and RNA 3' ends were dephosphorylated by PNK at pH6.5. Then pre-adenylated linker L3 (3' modification with biotin) was linked to 3' end of RNA at 16°C overnight. 5' end of RNA was labeled with [γ- 32P] ATP and eluted with 1x Nupage loading buffer (Invitrogen) at 70°C for 10 min. Protein-RNA complexes were eluted with 1xNuPAGE loading buffer. Complexes were separated on 4-12% NuPAGE Bis-Tris gel (Invitrogen), transferred to a nitrocellulose membrane and exposure to a Fuji film at -80℃. Protein-RNA complexes were digested with proteinase K and RNA were reverse transcribed by Superscript III reverse transcriptase (Invitrogen) with Rclip primers. cDNAs were separated on 6% TBE-urea gel, then three bands were cut at 120-200 nt (high), 85-120 nt (medium) and 70-85 nt (low). cDNAs were circularized by Circligase II (Epicentre). Single-stranded cDNAs were hybridized with cut_oligo, and then digested by BamHI. Linearized cDNAs were PCR amplified and purified on PAGEs to obtain iCLIP libraries. For total RNA and nascent RNA, 4sUDRB and ribo– RNA-seq libraries were prepared with Illumina TruSeq RNA Sample Prep Kit v2 (P/N 15026495) without oligo(dT) enrichment. Deep sequencing was performed on Illumina Hiseq 2500 at CAS-MPG Partner Institute for Computational Biology Omics Core, Shanghai, China. For iCLIP-seq, the protein-RNA complexes were treated with 5 Kunz U/ml of micrococcal nuclease with 5 μg yeast tRNAs for 5 min at 37°C, and RNA 3' ends were dephosphorylated by PNK at pH6.5. Then pre-adenylated linker L3 (3' modification with biotin) was linked to 3' end of RNA at 16°C overnight. 5' end of RNA was labeled with [γ-32P] ATP and eluted with 1x Nupage loading buffer (Invitrogen) at 70°C for 10 min. Complexes were separated on 4-12% NuPAGE Bis-Tris gel (Invitrogen), transferred to a nitrocellulose membrane and exposure to a Fuji film at -80℃. Protein-RNA complexes were digested with proteinase K, and RNA were reverse transcribed by Superscript III reverse transcriptase (Invitrogen) with Rclip primers. cDNAs were separated on 6% TBE-urea gel, then three bands were cut at 120-200 nt (high), 85-120 nt (medium) and 70-85 nt (low).cDNAs were circularized by Circligase II (Epicentre). Single-stranded cDNAs were hybridized with cut_oligo, and then digested by BamHI. Linearized cDNAs were PCR amplified and purified on PAGEs to obtain iCLIP libraries, which were subjected to Illumina HiSeq 2500 at CAS-MPG Partner Institute for Computational Biology Omics Core, Shanghai, China. RNA-Seq and RIP-seq
attributes.tag: GEO Accession
attributes.value: GSM2416840