GSM8160944: F002_hPSC-CMs_RAMP; Homo sapiens; OTHER
Total RNA was isolated using NZYol RNA Isolation Reagent (NZYTech) The library preparation of RNA-AMP-seq was performed using a similar procedure previously employed for IMPLICON (DOI: 10.1093/nar/gkaa567). Briefly, a first PCR amplifies each region per sample in individual reactions: 1ug (2μl) of cDNA is amplified with 2μl of each forward and reverse 10μM primer in 8μl of 2x KAPA HiFi Uracil+ ReadyMix in a final volume of 16μl. cDNA was amplified using the following conditions: initial denaturation at 95°C for 5min, 35 cycles of 98°C denaturation for 20 seconds, variable annealing temperature for 15 seconds and extension at 72°C for 60 seconds; followed by a final extension at 72°C for 10 minutes. All PCR reactions for each individual sample were pooled together and samples cleaned-up using 1.5x AMPure XP beads and eluted in 20μl H2O. Then a second PCR reaction, attached barcoded Illumina adapters to the pooled PCR samples ensuring that each sample pool receives a unique reverse barcoded adapter. The 20μl PCR pool was amplified using 1μl of 10μM Illlumina PE1.0 primer (same for all samples) and 1μl of 10μM Illumina iTAG primer (distinct for each sample) and 25μl of 2x KAPA HiFi Uracil+ ReadyMix in a 50μl volume reaction using the following conditions: initial denaturation at 98°C for 45 seconds, 5 cycles of 98°C denaturation for 15 seconds, 65°C annealing for 30 seconds and extension at 72°C for 30 seconds; followed by a final extension at 72°C for 5 minutes. Reactions were cleaned-up with 1x AMPure XP beads and eluted in 20μl. Finally, 2ug of each iTAGed sample are pooled together and cleaned-up with 1x AMPure XP beads and eluted in a final volume of 20μl. The library was verified by running 1:30 dilutions on an Agilent bioanalyzer Amplicon sequencing