GSM6531755: batch2_polya_seq_Fus_P423L_rep1; Mus musculus; RNA-Seq
RNA was purified by Trizol reagent (Sangon Biotech, Cat. #B610409) Binding oligo: /Index//iSp18/ACTCTGCGTTGATACCACTGCT/iBiodT/CCG/iSpC3/CGGAAGCAGTGGTATCAACGCAGAGTNNNNNNNNNNNN/Reverse complementary sequence of index//ideoxyU/TTTTTTTT/3InvdT/ was synthesized for the enrichment and ligation of poly(A) mRNA. /Index/ was designed as the 8 base pairs of sample index. /iSp18/ was synthesized as an internal 18-atom hexaethyleneglycol spacer. /iBiodT/ was biotin labeled dT. /iSpC3/ was a C3 Spacer designed to increase oligos' self-anneal. NNNNNNNNNNNN was 12 N as the UMI (unique molecular identifiers) for each sequencing read. /ideoxyU/ was used for USER enzyme digestion to remove the following oligo dT before sequencing. /3InvdT/ was 3' inverted dT which blocked the reverse transcription from undigested oligo. Low salt buffer (0.15 M NaCl, 20 mM Tris pH 7.5, and 1 mM EDTA) was prewarmed at 70 °C. Binding oligos were dissolved in Low salt buffer to make a final concentration of 8 μM. 10 μl of binding oligo was added to 20 μl Streptavidin Magnetic Beads (Thermo, Cat. #65001) which were washed with Washing/binding buffer (0.5 M NaCl, 20 mM Tris- pH 7.5, and 1 mM EDTA) twice. Samples were incubated at room temperature for 30 min with occasional agitation by hands. Beads were then washed with Washing/binding buffer twice. 5 μg total RNA, which was extracted by Trizol reagent (Sangon Biotech, Cat. #B610409) following the manufacturer's instructions, was dissolved in 50 μl of Washing/binding buffer, denatured at 65 °C for 5 min, and quickly chilled on ice for 2 min, before being incubated with 20 μl of beads. Samples were incubated at room temperature for 30 min and then washed with Washing/binding buffer twice. 0.8 μl 10 mM ATP, 4 μl T4 RNA ligase Reaction buffer (NEB, Cat. # B0216S), 8 μl 50% PEG8000, 1 μl T4 RNA ligase 2 (NEB, Cat. # M0239), 2 μl RNase inhibitor (NEB, Cat. # M0314S), and 4.2 μl H2O were added and incubated at room temperature for 2 h. Beads were washed with Washing/binding buffer twice， followed by 1X CutSmart buffer (NEB, Cat. # B7204) once. 49 μl 1X CutSmart buffer and 1 μl USER enzyme (NEB, Cat. # M5507) were mixed with the beads and incubated at 37 °C for 30 min. After being washed with pre-warmed 70 °C Low-salt buffer at room temperature for 2 min, beads were washed with H2O once. RT reaction was conducted by adding 4 μl 5X SuperScript IV RT Reaction Buffer (Thermo, Cat. #18091200), 1 μl 10 mM DTT, 1 μl 10 mM dNTP, 1 μl RNase inhibitor, 1 μl TSO oligo AAGCAGTGGTATCAACGCAGAGTACATrGrGrG. 3 μl 50% PEG8000, 1 μl SuperScript IV Reverse Transcriptase (Thermo, Cat. #18091200), and 8 μl H2O and incubating at 50 °C for 30 min. Beads were then washed with Low salt buffer and subsequent H2O once. Pre-PCR was conducted by using 25 μl NEBNext® High-Fidelity 2X PCR Master Mix (NEB, Cat. #M0541S), 1 μl 20 μM PCR oligo AAGCAGTGGTATCAACGCAGAGT, and 24 μl H2O at 98 °C for 3 min; 10 cycles of 98 °C for 10 s, 60 °C for 15 s, and 72 °C for 3 min; 72 °C for 5 min. Samples were purified by 0.9X of VAHTS DNA clean beads to 20 μl. The purified sample was used to construct the sequencing library for Sequel II Systems (PacBio) using SMRTbell Express Template Prep Kit 2.0 kit (PacBio) under the manufacturer's instructions.