GSM5014526: CRISPRi_ELAVL1_rep1; Homo sapiens; OTHER
RNA was extracted using Direct-zol-96 RNA Kit (Zymo). TLR7 RT-PCR was performed using Stratagene Brilliant II SYBR Green QRT-PCR Master Mix (50°C for 30 min, 95°C for 10 min, then 35 cycles of 95°C for 30 seconds, 60°C for 1 min, and 72°C for  30 seconds, finally 72°C for 5 min). The second PCR with Ad1_TLR7_F and Ad2_TLR7_R primers was performed using NEBNext Master Mix (2.5uL 1st PCR product with 500 nM primers in program: 98°C for 1 min, 20 cycles of 98°C for 30 seconds, 60°C for 30 seconds, and then 72°C for 40 seconds, finally 72°C for 5 min). The third PCR with indexed primers containing barcodes was performed using  NEBNext Master Mix (2uL of 1:10 diluted 2nd PCR product with 1uM indexed primers in program: 98°C for 1 min, 10 cycles of 98°C for 30 seconds, 72°C for 30 seconds, and then 72°C for 40 seconds, finally 72°C for 5 min). Finally, all the samples were pooled and purified using Zymo DNA clean & concentrator. The library was size-selected to 250-300 bp. The gel slices were cut and the DNA was recovered by Zymo Gel DNA recovery kit (Zymo Research). The concentration of the library was quantified by KAPA Library Quantification Kit for Illumina (Roche) and sequenced on an Illumina Miseq 2X75 cycle.