title: GSM6640340: Transfection of pXG-SRSF3 and pcDNA3-pri-mir-142_WT repeat3; Homo sapiens; OTHER
library_construction_protocol: Illumina adapter sequences were incorporated to the libraries using PCR. 1.5 μg of pXG-empty vector, pXG-SRSF7, or pXG-SRSF3 was co-tranfected with 1 μg of pcDNA3-pri-mir-142_WT into one well in the 6-well-plate of HCT116-DICER KO cells. The cells were also harvested two days later. All the total RNAs were extracted using TRIzol™ Reagent (Thermo Fisher, Cat. 15596018). We used iLIME-seq to clone and sequence pre-mir-142 from these total RNAs (Le et al., 2022). In short, 2.5 μg of total RNA was incubated with 16 units of T4 RNA ligase 1 (NEB, Cat. M0437M) in the ligation buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 16 units of SUPERase•In RNase inhibitor (Thermo Fisher, Cat. AM2694), and 20% PEG 8000. The mixtures were incubated at 25°C for 2 h. The ligated RNA products were then subjected to reverse transcription reactions using SuperScript™ IV Reverse Transcriptase at 50°C for 20 min. The resulting cDNAs were amplified the first time by pre-mir-142 sequence-specific primers and the second time by sequencing primers, both by Phusion Hot Start II DNA Polymerase (Thermo Fisher, Cat. F549L). The final DNA products from the second PCR were subjected to next-generation sequencing using the Illumina Novaseq 6000.