title: GSM4066537: 22RV1 miR-194_Replicate 1 [HITS-CLIP]; Homo sapiens; RIP-Seq
library_construction_protocol: After 24 h, transfected 22RV1 cells were rinsed once with ice-cold PBS and UV irradiated with 600 mJ/cm2, 254 nm, in ice-cold PBS using a UV Stratalinker-1800 (Agilent). Cells were collected by scraping, and cell pellets stored at -80°C as one pellet per 100mm plate. One pellet per CLIP IP was lysed in 500 μl of 1 X PXL (1 X PBS, 0.1% SDS, 0.5% deoxycholate, 0.5% Igepal) + EDTA-free Complete protease inhibitor cocktail (PIC; Roche) for 15 min on ice, followed by trituration through a 21G needle and syringe 5 times. DNA was digested with 20 μl RQ1 DNAse (Promega) at 37°C for 10 min on a Thermomixer (750 rpm, Eppendorf). RNA was partially digested with RNase 1 (ThermoFisher) by adding 5 μl of 1:40 diluted RNase 1 in 1 X PBS at 37°C for 5 min on a Thermomixer (750 rpm), then returned to ice. Lysates were centrifuged at 21,000 g for 20 min at 4°C and supernatant transferred to a fresh tube.AGO-RNA complexes were immunoprecipitated using mouse IgA2 monoclonal anti-Ago2 antibody 4F9(Ikeda et al 2006); hybridoma sourced from University of Florida ICBR) with a mouse IgA antibody (GeneTex S107) used as a control. Antibodies (8 μg) were conjugated to 20 μl protein L Dynabeads (ThermoFisher, 88849) in PBS-Tw (1 X PBS, 0.05% Tween-20) for 45 min and washed three times with 1 X PXL (1 X PBS, 0.1% SDS, 0.5% sodium deoxycholate, 0.5% Igepal) before resuspending the beads with 450 μl of prepared lysate and rotating for 2 hr at 4°C. Bound AGO-RNA complexes were washed twice each consecutively with ice cold 1 X PXL, 5 X PXL (5 X PBS, 0.1% SDS, 0.5% sodium deoxycholate, 0.5% Igepal), and 1 X PNK (50 mM Tris-Cl pH 7.5, 10 mM MgCl2, and 0.5% Igepal). Beads were first treated with T4 PNK (NEB, M0201L; 20 U in 80 μl reaction volume) in the absence of ATP (37°C, 850 rpm for 20 min) to dephosphorylate 3' RNA ends followed by washes with 1 X PNK, 5 X PXL, and two washes with 1 X PNK at 4°C. The 3' preadenylated linker (NEBNext 3'SR adaptor for Illumina; /5rApp/AGA TCG GAA GAG CAC ACG TCT /3AmMO/) was ligated to the RNA fragments on bead using T4 RNA ligase 2 truncated KQ (NEB M0373; 100 U in a 40 μl reaction volume, 12% PEG8000, 1x RNA ligase buffer, 0.125 μM adaptor) in the absence of ATP at 16°C, overnight with periodic mixing. Beads were washed consecutively with ice cold 1 X PXL, 5 X PXL, and twice with 1 X PNK. Bound RNAs were then labelled with P32 γ-ATP using T4 PNK, 20 min at 37°C, and washed as above. AGO-RNA complexes were eluted with 40 μl 1 X Bolt LDS sample buffer (ThermoFisher) + 1% β-mercaptoethanol at 70°C for 10 min on a Thermomixer (1200 rpm). Samples were separated through Bolt 8% Bis-tris Plus gels (ThermoFisher) using BOLT MOPS SDS running buffer at 200 V for 75 min. Complexes were then transferred to nitrocellulose (Schleicher&Schuell, BA-85) by wet transfer using 1 X BOLT transfer buffer with 10% methanol. Filters were placed on a phosphor screen and exposed using a Typhoon imager (GE). 115-160 kDa regions (corresponding to RNA tags > 30 nt) were excised from the nitrocellulose. RNA was extracted by proteinase K digestion (2 mg/mL proteinase K, 100 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM EDTA, 0.2% SDS) at 50°C for 60 min on a Thermomixer (1200 rpm) followed by extraction with acid phenol (ThermoFisher, AM9712) and precipitation with 1:1 isopropanol:ethanol. RNA was pelleted by centrifugation then separated on a 15% denaturing polyacrylamide gel (1:19 acrylamide, 1 X TBE, 7 M urea). The wet gel was wrapped in plastic wrap and exposed to a phosphor screen and imaged using a Typhoon. Gel slices were cut corresponding to the expected size of the cross-linked RNA eluted by the “crush and soak” method as previously described (Jensen and Darnell 2008). Reverse transcription, 5' linker ligation and amplification were performed essentially as previously described (Van Nostrand et al 2016) using SR-RT primer for reverse transcription (IDT, AGACGTGTGCTCTTCCGATCT) with SuperScript IV, and a custom synthesised 5' linker (IDT, 5'SRdeg /5Phos/NN NNN NNN NNG ATC GTC GGA CTG TAG AAC TCT GAA C/3SpC3/). Products were amplified for 20 cycles using a common forward primer (NEBNext SR primer for Illumina) and barcoded reverse primers for each sample (NEBNext Index primers for Illumina). PCR products were purified using Qiagen Qiaquick PCR purification kit, separated on an 8% acrylamide (29:1) TBE non- denaturing gel, stained with SYBR Gold nucleic acid gel stain (ThermoFisher) and imaged on a ChemiDoc (BioRad). Products corresponding to an insert size of ~30 – 70 nt were excised from the gel and extracted by the “crush and soak” method as previously described (Jensen and Darnell 2006). Library quality and quantity was assessed by Bioanalyzer (Agilent) and qPCR, pooled and sequenced on an Illumina NextSeq 500 (1 x 75bp).
attributes.tag: GEO Accession
attributes.value: GSM4066537