title: GSM5660925: PAR_TAK_2; Homo sapiens; RNA-Seq
library_construction_protocol: 1mg of polysomal lysates was resolved by ultracentrifugation on 10%-50% sucrose gradients. For each sample 20% volume of non-polysomal fractions from 40S to 80S peaks were pooled and spiked with a library of RNAs for normalization purposes across samples (ERCC spike-in mix, Thermo Fisher Scientific). 1μg of RNAs from two biological replicates from parental and LARP1 KO pools were depleted of rRNA with the Ribozero human, mouse, rat kit (Illumina) and  libraries for RNA-Seq were prepared. To identify the TSS of mRNAs protected by LARP1 with non-polysomes, 1 μg of the two parental RNA replicates was treated with alkalyne phosphates to blunt spourious 5' termini, and CIP-treated RNAs were decapped with Tobacco Alkaline Phoshpatase (TAP, Epicentre). Exposed 5' termini were ligated to an RNA oligo, then library were processed for RNA-Seq analysis.Differential analysis of non-polysomal transcripts upon TOR inhibition of parental / LARP1 KO cells. All libraries were sequenced on Illumina Novaseq 6000 platform in pair end mode resulting in 150 base length reads at Macrogen, Korea