title: GSM2975534: AGO1 PARCLIP replicate 2; Drosophila melanogaster; RIP-Seq
library_construction_protocol: For library preparation, cells were thawed on ice and quickly lysed in NP40 lysis buffer (50 mM Tris–HCl pH 7.4, 100 mM NaCl, 1 % Igepal CA-630 (NP40), 0.1 % SDS, 0.5 % sodium deoxycholate, Complete Protease Inhibitor to a final concentration of 2x, RNAsin 40 U/ml lysis buffer; 1 ml lysis buffer per approximately 0.3*109 cells, 1.2*109 to 1.3*109 cells in total per sample). After treatment with RNaseI (1:333 v/v or 300 U/ml lysate for HITSCLIP and 1:400 v/v or 250 U/ml lysate for PARCLIP samples, because of slight concentration differences between lysate; TurboDNase (4 U/ml) for 3 min at 37 °C and 1100 rpm, immunoprecipitation (IP) was carried out with polyclonal AGO1 (Abcam #ab5070, 20 µg per sample) coated magnetic protein A dynabeads (Life Technologies #10002D)(100 µl) on spin-cleared cell extracts for 2 hrs at 4 ºC.After IP, CLIP samples were stringently washed three times with high salt buffer (50 mM Tris–HCl, pH 7.4, 0.666 M NaCl, 1mM EDTA, 1% Igepal CA-630 (NP40), 0.1% SDS, 0.5% sodium deoxycholate), followed by PNK-buffer washes. Samples were radioactively 5'end-labelled with γ-32P-ATP including a subsequent addition of 1 µl high-molar ATP to the reaction for efficient 5'end phosphorylation. The crosslinked protein-RNA complexes were resolved on a 4-12 % Bis-Tris-Polyacrylamid gel. The SDS-PAGE gel was transferred to a nitrocellulose membrane and the protein-RNA complexes migrating at an expected molecular weight were excised. RNA was isolated by Proteinase K treatment and phenol-chloroform extraction, ligated to 3' adapter and 5' adapter, reverse transcribed using Superscript III (Life Technologies #18080044), PCR-amplified (PCR cycles: HITSCLIP 20 cycles; PARCLIP 19 cycles), and gel-purified. Note, after the 3'Adpater ligation step, each AGO1 sample was split into approximately 19-24 nt (miRNA fraction) and 24-35nt (target fraction) long fragments, cloned, amplified and sequenced separately. The amplicons were sequenced single-end as a multiplexed pool on HiSeq2000 (Illumina). Sequencing Reads of 19-24 nt and 24-35nt fractions were combined.
attributes.tag: GEO Accession
attributes.value: GSM2975534