GSM5287859: 5S_rep2_invivo_DMSP; Homo sapiens; OTHER
For SHAPE-MaP, 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 μL (200 U/μL) of SuperScript II (Invitrogen), 6 mM Mn2+ and gene-specific primers for SLERT(5S rRNA was used as a spike-in control). Mn2+ was removed using G-25 micro-spin columns (GE Healthcare) after SHAPE-MaP reverse transcription. Second-strand synthesis was performed with Q5 hot start high-fidelity DNA polymerase and nested PCR was performed to further improve DNA yield. SHAPE-MaP libraries were prepared from 1 ng of DNAs for SLERT and 5S rRNA, 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 X Ten at CAS Key Laboratory of Computational Biology Omics Core, Shanghai, China.