title: GSM4221401: 106a_original_substrate; synthetic construct; OTHER
library_construction_protocol: Illumina adapter sequences were incorporated to the libraries using PCR. To synthesize each group of pri-mir-106a, pri-mir-342 or pri-mir-514a-1, two oligos each containing 3-nt randomized sequence were annealed using the complementary region of 20 base pairs and then annealed DNA was extended using Klenow (Thermo Scientific, EP0421). Subsequently, the Klenow-extended DNA duplexes were amplified by two PCR primers in which the forward primers contain the T7 promoter sequence. 200 ng of each dsDNA group was used in a 20 µl in vitro transcription reaction using the MEGAscript T7 transcription kit (Invitrogen, AM13345).  In the high-throughput pri-mir-106a processing assays, 5 pmol of each pri-mir-106a group was incubated with 10 pmol of the D3-G2 complex in 10 µl standard reaction buffer at 37°C for 120 min. The original substrate from the control samples and the cleaved product were the gel-purified. The control sample was reverse transcribed using the R-RA3-6N-106a primer and Superscript IV reverse transcriptase. The synthesized cDNAs were first amplified using F-RA5-106a and RPI4 (index:TGGTCA) primers. Then the first PCR product was further amplified using RP1 and RPI4 primers to get the final PCR product for NGS. The cleaved samples (i.e., F2 fragment) were first ligated to the 3'-adapter (4N-RA3) using T4 RNA ligase 2, truncated KQ(NEB, M0373L).The RA3-ligated cleaved samples were separated from the unligated RNAs and 4N-RA3 by 10% Urea-PAGE and then gel-purified. The RA3-ligated RNAs were further ligated to the 5'-adapter (RA5-4N) using T4 RNA ligase I (NEB, M0204L). These two-adapter ligated RNAs were then reverse transcribed using the R-RA3 primer and Superscript IV reverse transcriptase and the synthesized cDNAs were amplified using RP1 and RPIx primers. The high-throughput pri-miRNA processing assays were carried out similarly for pri-mir-514a-1 and pri-mir-342 variants using the D3TN1-G2 complex. The pooled control and cleaved RNAs were first ligated to the 3'-adapter (4N-RA3) using T4 RNA ligase 2, truncated KQ. After that, the 4N-RA3-ligated control RNAs were further processed similarly as what described for arti1. The 4N-RA3-ligated cleaved RNAs were subsequently cloned similarly as what described for the pri-mir-106a library. The final PCR products were subjected to Illumina Nextseq 500 sequencer using NSQ 500/550 High Output KT v2.5 (150 CYS) (Illumina, 20024907) in single-end running mode.
identifiers.namespace: GEO
identifiers.id: GSM4221401