GSM1525046: RIP_Replicate_2; Homo sapiens; RIP-Seq
Growth media was collected, and cells were washed twice with 10 mL of room temperature PBS (Gibco). Cells were crosslinked by incubation in 0.1% (v/v) formaldehyde in PBS for 10 minutes at room temperature, under very gentle agitation. Crosslinking was quenched by the addition of Glycine to 133 mM and gentle agitation for an additional five minutes at room temperature. The liquid phase was then aspirated; cells were washed twice with room temperature PBS, harvested by scraping, allotted into samples of 1×10^7 cells (typically three samples per 10 cm dish), and pelleted at 200 g in a swinging bucket rotor. PBS was aspirated and cell pellets were flash-frozen in liquid nitrogen and stored at –80°C until use. Cell pellets were thawed on ice, resuspended into 1 mL of ice-cold RIPA(+) buffer (standard RIPA supplemented with 0.1 U/uL RNAseOUT (Life Technologies), 1x EDTA-free Proteinase Inhibitor Cocktail (Thermo Scientific) and 0.5 mM DTT), and lysed for 10 minutes at 4°C with end-over-end agitation. Samples were then sheared using a Branson Digital Sonifier 250 (Emerson Industrial Automation) at 10% amplitude for three 30-second intervals (0.7 seconds on + 1.3 seconds off), with 30-second resting steps between intervals.  Samples were held in ice-cold metal thermal blocks throughout sonication.  Sheared samples were then clarified by ultracentrifugation and diluted with 1 mL each of ice-cold Native Lysis Buffer(+) (25 mM Tris, pH 7.4, 150 mM KCl, 5 mM ETA, 0.5% (v/v) NP-40, supplemented with inhibitors and DTT, as above), filtered through a 0.45 um syringe-mounted filter, and flash-frozen in liquid nitrogen before use. Clarified lysates were thawed on ice and pre-cleared by incubation with buffer-equilibrated magnetic Protein G beads (Life Technologies) for 30 minutes at 4°C, with end-over-end rotation. Cleared lysates corresponding to 5×10^6 cells were then incubated with 6 ug rabbit anti-FLAG antibody (SIGMA) or Rabbit normal IgG (Cell Signaling Technology), for two hours at 4°C with end-over-end rotation. Buffer-equilibrated magnetic Protein G beads were then added and the samples were again rotated end-over-end for one hour at 4°C. Resin was collected and twice washed with Native Lysis Buffer(+) for 10 minutes at 4°C, with end-over-end rotation. Beads were suspended in 56 μL nuclease-free water, and processed alongside input samples (56 μL; 5.6% of the total). All samples were brought to 100 μL in 1x PBS, 2% N-Lauroyl Sarcosine, 10 mM EDTA, 5 mM DTT, 0.4 U/μL RNaseOUT and 2 mg/mL proteinase K (Ambion), final. Formaldehyde crosslinks were reversed by incubation at 42°C for one hour, and then 55°C for one hour, in a thermocycler. RNA was purified using four volumes (400 μL) Agencourt RNAClean XP Beads (Beckman Coulter), following the manufacturer’s protocol, and eluted into 30 μL nuclease-free water.  Residual DNA was removed by treatment with 5 U RNase-free DNAs (RQ1, Promega) in 50 μL, following the manufacturer’s protocol. RNA was subsequently purified using four volumes (200 μL) Agencourt RNAClean XP beads, eluted into 20 μL nuclease-free water. Sample 1. Five ng of template DNA was PCR amplified using Pfu Ultra II HS polymerase (Agilent), according to the manufacterer's protocol, using gene-spectific primers appended with standard Illumina adapters and multiplexing indexes.  The reaction volume was 50 uL; PCR was performed over 19 cycles. Samples 2–7. Twenty ng of RNA was reverse-transcribed using SuperScript III Reverse-transcriptase (Life Technologies) and a gene specific primer, in 20 uL, following the manufacturer's protocol. cDNA was then amplified over 26 cycles of PCR, in 200 uL reactions, using primers as described for Sample 1. All libraries were putified twice with Agencourt RNAClean XP beads, and eluted into 30 μL nuclease-free water.