GSM7109828: High_salt_precipitated_RNA; Homo sapiens; RNA-Seq
For ribosome IP experiments, 24 hrs after the second transfection of siRNAs, 4 million cells were resuspended in 1 ml of IP buffer (50 mM Tris-HCl, pH7.5, 100 mM KCl, 12 mM MgCl2, 1 % NP40, 1 mM DTT, 100 unit/ml RNase inhibitor (Ambion), 1× protease inhibitor cocktail (Roche) and 100 𝛍g/ml cycloheximide). Cells were incubated on ice for 40 minutes with occasional inversion and centrifuged at 12,000g for 15 minutes at 4ºC. Fifty microliters of the cleared lysate were saved before combining the rest with 2.5 𝛍g of Y10b anti-5.8S RNA antibody immobilized on Protein G Dynabeads. The lysate-Dynabeads complex was rotated for 3 hrs at 4ºC, washed five times with high salt buffer (IP buffer with 300 mM KCl), and resuspended in Trizol for mRNA-seq analysis. For fractionation of RNA condensates, 2 million cells were resuspended in 250 𝛍l of fractionation buffer (50 mM Tris-HCl, pH7.6, 50 or 200 mM KCl, 5 mM MgCl2, 0.1 % NP40, 1 mM DTT, 100 unit/ml RNase inhibitor (NEB, #M0314), 1× protease inhibitor cocktail), incubated on ice for 10 minutes, and centrifuged at 2,000g for 2 minutes to remove nuclei. Cell lysates were transferred to new microfuge tubes and centrifuged again. After 40 microliters of the lysate were taken out for the total cytoplasmic lysate, 200 microliters of the lysate were centrifuged at 12,000g for 15 minutes. The upper fraction was saved as the supernatant. The pellet was washed twice with the fractionation buffer and resuspended in Trizol for RNA or 8M urea for protein analysis. RNA integrity was confirmed by a bioanalyzer using an Agilent RNA 6000 Pico Kit (Agilent, Santa Clara, CA). The isolated total RNA was processed for preparing mRNA sequencing library using TruSeq stranded mRNA sample preparation kit (illumina, San Diego, CA) according to manufacturer's instruction. Simply, mRNAs were isolated from 400 ng total RNA by RNA purification bead using polyA capture, and followed by enzyme shearing. After first and second strand cDNA synthesis, A-tailing and end repair were performed for ligation of proprietary primers that incorporate unique sequencing adaptors with index for tracking illumina reads from multiplexed samples run on a single sequencing lane. For each library, an insert size of approximately 200 bp was confirmed by a bioanalyzer using an Agilent DNA  Kit (Agilent, Santa Clara, CA) and quantification of library was measured by real-time PCR using CFX96 real time system (BioRad, Hercules, CA). All the samples were sequenced on Illumina NextSeq 500 Sequencer with 75-bp Paired end High Output Kit. The raw image data was transformed by base-calling into sequence data and stored in FASTQ format.