GSM2676358: V1_ADAR_KD_replicate2; Homo sapiens; OTHER
Total RNA from HepG2 cells was isolated using Trizol (Invitrogen) and treated with DNase I (Invitrogen). Poly-A mRNA was isolated using mRNA Sequencing Sample Preparation kit (Illumina Proprietary). Random-primed cDNA synthesis was done on 2μg of total RNA using M-MLV reverse transcriptase (Invitrogen). qPCRs for ADAR were done as described. Analysis was performed by the SDS 2.3 software using the absolute quantification method with ABL for the normalization. All reactions were run on ABI 7900HT genetic analyzer (Applied Biosystems). Probing the mRNA with S1 nuclease (digest at un-paired bases) and RNase V1 (digest at paired bases) was done as described in Kertesz et al. (2010). Libraries were prepared as described in the PARS-seq protocol, using the TruSeq Small RNA sample preparation kit of the Illumina. We ligated the 5' adapter as described in the TruSeq Small RNA sample preparation protocol (Illumina) and stopped the reaction with 1µl stop solution (a component of the TruSeq Small RNA sample preparation kit) in 28C for 15 min. 3' end treatment with Antarctic phosphatase was done as described in the directional mRNA-Seq sample preparation protocol, followed by its inactivation with ethanol precipitation. 3' adapter ligation, reverse transcription and PCR amplification were performed as described in the TruSeq Small RNA sample preparation protocol. The cDNA was size selected and cleaned using E-Gel 4% agarose (Invitrogen) and Agencourt AMPure XP beads (Beckman Coulter). The library was quantified using Qubit (Invitrogen) and validated using Agilent 2100 Bioanalyzer. Libraries were sequenced on Illumina Hiseq2500 machine.
GEO Accession
GSM2676358