GSM7883525: Dicer-R1855A double cleavage replicate 1; synthetic RNA; RNA-Seq
We first ligated the control RNA and single cleavage products to the 4N-RA3 adapter using T4 RNA Ligase 2, truncated KQ enzyme (NEB, M0373L). The resulting 4N-RA3-ligated RNA were then gel-purified. Next, we mixed the purified RNAs in a reverse transcription (RT) mixture containing cirRTP primer and Superscript IV Reverse Transcriptase (Invitrogen) and incubated the mixture for 60 min at 50°C. To degrade the RNAs, we added 0.1 M NaOH to the RT mixture and incubated it at 98°C for 10 min. We then purified the resulting cDNAs and circularized them using CircLigase ssDNA ligase (Epicentre). After separating the circularized cDNAs from linear cDNAs in an 18% urea-PAGE gel and gel-purifying them, we amplified the purified circularized DNAs by PCR using RP1 and RPI1 and one of the RPIx primers, respectively. We used the RP1 and RPIx primer systems from the Truseq Illumina primers. For the double cleavage (DC) fragments, we ligated them to the 4N-RA3 adapter and separated the resulting 4N-RA3-ligated DC from unligated DC and free 4N-RA3 in a 12% Urea-PAGE gel, which we then gel-purified. We ligated the purified 4N-RA3-ligated DC with the 4N-RA5 primer using T4 RNA ligase 1, and reverse-transcribed the double-ligated DC using Superscript IV Reverse Transcriptase and RTP primer. Finally, we amplified the resulting cDNA by PCR using RP1 and RPIx. adapter ligation, circularization