GSM6415294: Opto_0_min; Homo sapiens; RNA-Seq
H4 cells carrying Cry2Olig-PKR cells were plated on fibronectin treated 96-well glass-bottom plate. Cells were illuminated according to experimental set-up using the optoPlate device set to continuous illumination intensity of 7.5 mcd as mentioned in Optogenetic stimulation. Cells were immediately lysed using 120 µL Trizol (Gibco, 15596026) per well, under red light, and incubation for lysis for 5 min at room temperature. RNA was extracted according to the manufacturer's protocol. RNAseq libraries were prepared following an adaptation of the single-cell method “CEL-Seq2” where we treated every illumination duration as a single cell (Hashimshony et al., 2016; Wangsanuwat et al., 2020). Briefly, sample RNA was reverse transcribed with Cel-Seq2 primer #4 (25 ng) using Superscript II reverse transcriptase. Second Strand Synthesis (SSS) was carried out via E. coli ligase (Invitrogen, Cat. # 18052–019) and DNA polymerase I (Invitrogen, Cat. # 18010–025) in SS buffer for 2 hours at 16°C. Next, cDNA was purified with 1:1 AMPure XP DNA beads (Beckman Coulter, Cat. # A63881), and washed with 80% ethanol twice, before elution into nuclease-free water and vacufuge concentrated. In vitro transcription was then carried out using MEGAscript T7 kit (Thermo Fisher Scientific, AMB13345) for 13 hours at 37°C, followed by RNA treatment using EXO-SAP (Thermo Fisher Scientific, 78200.200.UL) for 15 min at 37°C, and finally the samples were fragmented (200 mM Tris-acetate (pH 8.1), 500 mM KOAc, 150 mM MgOAc) at 94°C for 3 min before being quenched by 0.5 M EDTA. The fragmented RNA was size selected with 0.8x AMPure RNA beads (RNAClean XP Kit, Beckman Coulter, Cat. # A63987) and eluted in nuclease-free water.