title: GSM8368398: wild type sw480 colorectal cancer cell, RIP input RNA; Homo sapiens; RIP-Seq
library_construction_protocol: Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. Collect enough tissue culture cells, this is comparable to 10000000 cells per RIP. Resuspend final cell pellet with an equal volume of polysome lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH 7.2), 0.5% NP40, 1 mM DTT, RNase inhibitors, protease inhibitors)，incubated on ice for 5 minutes and cellular debris was pelleted by 4°C centrifugation at 15,000 x g for 15 minutes. After centrifugation 40μL of supernatant was collected for RNA extraction as input sample. The remaining supernatant was used for immunoprecipitation.100ul protein A/G magnetic beads were washed with NT2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2 and 0.05% NP-40) three times. Magnetic beads resuspended in 850 μl of the immunoprecipitation reaction solution (200 units of an RNase inhibitor, 400 μM RVC, 10 μl of 100 mM DTT, 30 μl of 0.5 mM EDTA and 800 μl of cold NT2 buffer). Cleared cell lysates were incubated with immunoprecipitation reaction solution at 4 °C for 4 h on a tube rotator. The beads were then washed with 1 ml of ice-cold NT2 buffer five times.TRIzol was added to input and immunoprecipitation (IP) samples to isolate total RNA. RNA quality control was measured on a Qubit. Sample libraries were prepared with SMART-Seq v4 Ultra Low Input RNA Kit (Takara Clontech Kit Cat# 63488). Libraries were sequenced on a NoveSeq 6000 (Illumina) . RNA Immunoprecipitation sequencing