MinION sequencing; Quantitative measurement of full-length transcript isoforms reveals precise regulation of splicing in human heart cells
Quantitative measurement of full-length transcript isoforms reveals precise regulation of splicing in human heart cells
iPSCs were differentiated into cardiomyocytes as a monolayer through the modulation of WNT signaling as previously described.  iPSCs were plated at low density on Matrigel coated plates to have them 70-80% confluent after 4 days (Day 1 of differentiation). Differentiation was induced with 3ml of RPMI 1640 (Life Technologies 11875-093) with 1X B27® Minus insulin (Life Technologies 0050129SA), supplemented with 6μM CHIR (TOCRIS 4953). On day 6 media was replaced with 3ml of RPMI 1640 with 1X B27® Minus Insulin, supplemented with 5μM IWR. On day 6 media was replaced with 3ml of RPMI 1640, 1X B27® Minus Insulin. From day 8 cells were kept in RPMI 1640 (Life Technologies 11875-093) with 1X B27® Serum-Free Supplement (Life Technologies 17504-044).  From day 12 to day 15 cells were treated with RPMI 1640 no Glucose (Life Technologies 11879-020) with 1X B27.  Then cells were allowed to recover for 2 days in RPMI 1640 with 1X B27 and subsequently replanted at a density of 3Mi cells/well. Cells were then maintained in RPMI 1640 with 1X B27 for 4 more weeks before being collected for experiment.    RNAs were cleaned up and treated with DNase using ZymoResearch RNA Clean and Concentrator-5.  Reverse transcription   A total of 4.5 µL mixture containing 5 ng of purified iPSC-CM RNAs with 2.8% ERCC and 3% Sequins Version A, 1µL of 10µM oligodT (/5SpC3/A*A*G*CAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN), 1µL of 10mM dNTP, 0.1µL of Takara recombinant ribonuclease inhibitor (40U/µL) were incubated at 72 ˚C for 3min, 4˚C for 10min and 25˚C for 1min. Another 5.5µL mixture containing 2 µL of 5x SuperScript II buffer, 2µL of 5M betaine, 0.5 µL of 100 mM DTT, 0.5 µL of SuperScript II (200 U/µL), 0.25 µL of Takara recombinant ribonuclease inhibitor, 0.1 µL of 100 µM TSO, 0.09µL of nuclease-free water, 0.06 µL of 1M MgCl2 were added. The total 10 µL RT reaction were incubated at 42˚C for 90 min, 10 cycles of 50˚C for 2min and 42˚C for 2 min4. The reaction were then stopped by incubation at 70˚C for 15 min and hold at 4˚C. The reaction were treated with 1µL of 1:10 dilution of NEB RecJf (Catalog # M0264S) at 37˚C for 30min and 65˚C for 20min.   PCR amplification of full-length cDNA  25µL of 2x NEB Q5 HotStart master mix, 0.25 µL of 10µM PCR primers (ONT_Index1_ISPCR and ONT_Index2_ISPCR)5 and 14.5 µL of nuclease-free water were added to the RT reaction. The PCR were performed as follows: 98˚C for 30s, 20 cycles of 98˚C for 10s, 67˚C for 15s, and 72˚C for 6min, then 72˚C for 2min, and hold at 4˚C. PCR reactions were purified using 0.75 volume of Ampure XP beads and eluted to 25 µL of nuclease-free water.
Genetics Department, Stanford University
E-MTAB-7334:WT_A2B_run2_s