GSM4007376: Excitatory neurons, Scramble 1; Homo sapiens; RNA-Seq
After 60 days of culturing after infection, we aspirated all the media and washed the cells with PBS before freezing them at -80C for later RNA extraction. We extracted RNA from shRNA-infected, human ESC-derived neurons in a 12-well plate. We lysed the neurons in the tissue-culture plate with TRIzol Reagent (ThermoFisher Scientific) and immediately transferred the lysate to microfuge tubes for trituration. We then isolated the RNA by chloroform phase separation, precipitation with 2-propanol, washing with 75% ethanol, and eluting in water. We reverse transcribed 1 ug of total RNA with the partial P7 adapter (Illumina_4N_21T) and dNTPs with the addition of spiked-in azido-nucleotides (AzVTPs) at 5:1. We click-ligated the p5 adapter (IDT) to the 5′ end of the cDNA with CuAAC. We then amplified the cDNA for 21 cycles with Universal primer and 3′ indexing primer and purified it on a 2% agarose gel by extracting amplicon from 200-300 base pairs. We pooled the libraries and sequenced single-end, 75 base-pair reads on a Nextseq 550 (Illumina).