GSM3763005: Total RPF; Homo sapiens; OTHER
The RNA were extracted with TRIzol reagent(thermo ambion, REF,15596018) and its standard protocol. After clarification, 5 OD260 units of lysates were treated with 450 units of RNaseI (Ambion) for 45 min at RT. RPFs were isolated with Sephacryl S400 (GE, MicroSpin S-400 HR, 27514001). mRNAs and RPFs were isolated with TRIzol reagent(thermo ambion, REF,15596018). The rRNA removal for total mRNAs and RPFs was accomplished using Ribo-zero Gold (rRNA removal reagent(Human/Mouse/Rat)(illumina, REF, RZG1224) & Core kit(illumina, REF, MRZ11124C). For RPF, the extracted RNA was size-selected from 15% denaturing PAGE gels, cutting between 26-34 nt. An oligonucleotide adaptor was ligated to the 3' end of footprints. Followed by reverse transcription, circularization and PCR amplification.