title: GSM6805302: HEK293T_15.5K_RIP_PEN_SHAPE_MaP_DMSO_rep2; Homo sapiens; OTHER
library_construction_protocol: Cell lysate was prepared and incubated with anti-FLAG. Afterwards, the RNA-protein complexes were immunoprecipitated with Dynabeads protein G. Then the on-beads modification was performed with SHAPE reagent NAI-N3 or DMSO (negative control) with the SHAPE modification buffer (100 mM HEPES, pH8.0, 6 mM MgCl2, 150 mM NaCl). Thereafter, RNA fraction was extracted using RNAzol reagent. After RNA isolation and DNase I-treatment, the 15.5K-interacted RNAs were ligated to 3' and 5' randomized RNA adaptors. Then the ligated products were subjected to rRNA, snRNA and some high-abundance snoRNA depletion utilizing an RNase H-based strategy. Following the reverse transcription under SHAPE-MaP conditions, the libraries were amplified with Illumina small RNA indexed PCR primers.