GSM5065623: NPOP_R1HeLa; Homo sapiens; OTHER
Following the trizol lysis, interphase pellet was subjected to RNase A/T1 (thermo scientific) degradation in RNase buffer (10 mM Tris-HCl, pH 7.5, 300 mM NaCl and 5 mM EDTA, pH 7.5). 2 µg of RNase A/T1 mix was added to the interphase pellet, mixed by pipetting and incubated at 37° C for 15 min. Interphase-RNase mixture was resolubilized in trizol reagent to recover the RNA-protein complexes. After addition of chloroform, tubes were incubated at room temperature for 5 min, vortexed and centrifuged at 12000 g for 10 min. This results in protein bound RNA at the interphase, unbound RNA in the aqueous phase while the unbound proteins in the organic phase. The aqueous and organic layers were discarded as described previously. Interphase pellet was precipitated in 1 ml methanol and spun down to remove the methanol. Next, the interphase was mixed with Proteinase K in appropriate buffer (0.1M NaCl, 10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.5% SDS and 200 µg/ml proteinase K). Samples were incubated at 50° C for 2 hours. After proteinase K digestion, free RNA was recovered from the aqueous layer by trizol extraction as described previously.  Samples were cooled and the released RNA was purified by standard trizol extraction. The upper aqueous layer containing the free RNA fragments (previously bound to proteins) was transferred to new tube and precipitated with isopropanol as per the manufacturer instructions.   Protein bound RNA's concentration was estimated using Nanodrop and up to 1 µg of RNA was incubated with DNase I, 1U (thermo scientific) at 37° C for 30 min to remove any traces of DNA contamination. 1 µl of 50 mM EDTA was added to the reaction mixture and incubated at 65° C for 10 min to terminate the reaction. RNA was purified using conventional trizol extraction from the aqueous layer. At this point, r-RNA depletion was performed with 1 µg input RNA using Ribo-cop kit (Lexogen) as per manufacturer instructions. Further, the ends of r-RNA depleted RNA were modified by treating with Calf intestine alkaline phosphatase (CIAP, Invitrogen) and T4 polynucleotide kinase (T4 PNK, thermo scientific) as per manufacturer protocol.  The end modification enabled the library preparation of these RNA fragments. RNA purity and concentration were assessed at each step using Nanodrop (thermo scientific) based on the absorbance ratio 260/280 >2. RNA integrity was evaluated using Agilent 2100 bioanalyser system. At least 50 ng of r-RNA depleted POP-seq RNA was used to generate sequencing libraries using the True-seq small RNA library prep kit (Illumina). All libraries were barcoded and sequenced in parallel on a Next-seq platform for 400 million reads to obtain 75 bp single end reads. POP-seq libraries prepared using Illumina True seq small RNA library preparation kit