title: GSM5897025: DC-KD4-F4R4; Homo sapiens; OTHER
library_construction_protocol: The Tet inducible stable URB1-KD HeLa cell lines were seeded and induced with 1ug/mL doxycycline on 6 cm dishes for 6 days. After washed with DPBS, cells were incubated with 270 ?L of cell culture medium and 30 ?L of 10x SHAPE Chemical in DMSO with the final concentration of NAI (EMD Millipore) at 100 mM for 10 minutes at 37 °C. After removed reaction medium, RNAs were isolated by 1 mL Trizol reagent (Invitrogen) according to the manufacturer?s protocol. The same procedure was also performed in parallel for the untreated control samples, but with the addition of only DMSO. In the denaturing control (DC) reaction, RNAs were suspended in a denaturing buffer containing 50% formamide and were incubated at 95 °C before modification with SHAPE reagents. For DMS samples, after washed with DPBS, cells were treated with the addition of 15uL DMS and incubation at 37°C for 5min. Cell culture media with DMS was slowly decanted. Then cells were washed twice in 30% v/v BME (diluted in DPBS) and collected into a 15ml centrifuge tube using a scraper. Centrifuge cells at 1000 × g at 4°C for 3min and decant the BME solution. Wash the cells with 1ml DPBS twice and centrifuge the cells again. Isolated RNAs were treated with DNase I (Ambion, DNA-freeTM kit) to remove possible DNA contamination. About 50-100 ng of RNAs were obtained under each treatment and were then used for SHAPE-MaP reverse transcription by adding 1 mL (200 U/mL) of SuperScript II (Invitrogen), 6 mM Mn2+ and gene-specific primers for 3' ETS. Second-strand synthesis was performed with TAQ DNA polymerase. The resulting PCR products were further isolated. Primers for SHAPE-MaP reverse transcription and second-strand synthesis (1st round PCR and nested PCR reactions) were listed in Supplementary Table S1. SHAPE-MaP libraries were prepared from 1 ng of DNAs, and size-selected with AmpureXP beads (Agencourt) with a 1:1 (bead to sample) ratio to obtain library DNA products spanning 100-400 bp in length. Final libraries were quantified using Agilent Bioanalyzer 2100 and QuBit high-sensitivity dsDNA assay. Deep sequencing was performed by Illumina NextSeq 500 at Shanghai Institute Nutrition and Health, Big Data Center Omics Core, Shanghai, China. About 15-25 million mapped sequencing reads were obtained for each sample, with 88% of bases at or above Q30. Isolated RNAs were treated with DNase I (Ambion, DNA-freeTM kit) to remove possible DNA contamination. About 50-100 ng of RNAs were obtained under each treatment and were then used for SHAPE-MaP reverse transcription by adding 1 mL (200 U/mL) of SuperScript II (Invitrogen), 6 mM Mn2+ and gene-specific primers for 3' ETS. Second-strand synthesis was performed with TAQ DNA polymerase. The resulting PCR products were further isolated. Primers for SHAPE-MaP reverse transcription and second-strand synthesis (1st round PCR and nested PCR reactions) were listed in Supplementary Table S1. SHAPE-MaP libraries were prepared from 1 ng of DNAs, and size-selected with AmpureXP beads (Agencourt) with a 1:1 (bead to sample) ratio to obtain library DNA products spanning 100-400 bp in length. Final libraries were quantified using Agilent Bioanalyzer 2100 and QuBit high-sensitivity dsDNA assay. Deep sequencing was performed by Illumina NextSeq 500 at Shanghai Institute Nutrition and Health, Big Data Center Omics Core, Shanghai, China. About 15-25 million mapped sequencing reads were obtained for each sample, with 88% of bases at or above Q30.