Instrument: Illumina MiSeq
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: Forty-eight hours post-transfection, total RNA were purified from cultured cells using RNeasy Mini Kit (Qiagen), as per the manufacturer's instructions. For each sample, a multiplex RT-PCR assay was first applied to simultaneously amplify multi-exonic regions within 108 genes in a single reaction. The multiplex RT-PCR reaction was carried out in 96-well plates using the OneStep RT-PCR kit (Qiagen) as recommended by the manufacturer, with the following changes: reactions were performed in a volume of 20 uL with 2 uL of the purified total RNA as input, and a mixture of 50 pairs of primers was added to each reaction with a final concentration of 0.25 uM for each individual forward and reverse primer. Thermocycler settings were: 50°C for 30 minutes, 95°C for 15 minute, 30 cycles of 94°C for 40 seconds, 58°C for 1 minute (slow ramp rate), 72°C for 3 minutes, and a final extension step at 72°C for 10 minutes. Two sets of barcodes were incorporated into forward and reverse primers, respectively, after the universal adaptors and added to the amplicons in the second PCR reaction to allow demultiplexing all reads that were derived from a particular treatment. Primers sequences were: forward primer, AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT (N denotes i5 barcode); reverse primer, CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (N denotes i7 primer). The second PCR was performed using Phusion High-Fidelity DNA Polymerase (Thermo Scientific), as per the manufacturer's instructions. For each 20 uL of reaction, 1 uL of the multiplex RT-PCR reaction products was used as templates. The thermal cycling conditions were as follows: 98°C for 30 seconds, 15 cycles of 98°C for 10 seconds, 65°C for 30 seconds, 72°C for 30 seconds, and a final extension step at 72°C for 5 minutes. The resulting libraries were pooled and sequenced.