GSM9081227: Hpb_RNA-seq_iPrimer-seq; Mus musculus; RNA-Seq
Directly lysis the cell by Digitonin The isolated RNA was eluted with the 20 μL reverse transcription mix consisting of 100 unit Hiscript III Reverse Transcriptase (Vazyme, R303), 40 unit RNase Inhibitor (Vazyme, R301),1 μL 25 mM dNTP mix, 1 μL 100 mM DTT, 1 μL 1 val Betaine (Sigma, B0300-1VL), 0.5 μL 5% Digitonin, 0.5 μL TSO-LNA and 0.5 μL 5 μM Probe. The reaction was incubated at 42℃ for 90 min. Following cDNA synthesis, the sample were pooled, cleaned and concentrated with cleanup beads at a 1:0.8 of DNA to beads ration and eluted in 30 μL of ddH2O. Second-strand synthesis and pre-amplification were performed in a 50 μL reaction, consisting of 2x KAPA HiFi Ready mix and 0.5 μM TSO-PCR primer, with the following PCR setup initial denaturation at 98 °C for 3 min, (denaturation at 98 °C for 15 s, annealing at 65 °C for 30 s, elongation at 72 °C for 6 min) repeated for 8 cycle, and a final elongation at 72 °C for 10 min. The DNA was cleanup using DNA cleanup beads at a ratio of 1:0.8 of DNA to beads and eluted with 30 μL of ddH2O. The library were prepared with the TruePrep DNA Library Prep Kit V2 for Illumina according to the manufacturer. Probe was designed by TSO, sample barcode(7-mer),UMI(8-mer) and Oligo(dT).