title: GSM9295175: Normoxia_AsPC1_Day7_rep3; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were lysed directly in well with TriReagent and extracted using the Zymo Directzol micro kit according to manufacturer protocols To generate RNA-seq lite libraries, NEB's Template Switching RT Enzyme mix was used (NEB Cat# M0466L). 250 ng of total RNA from each sample was brought to a final volume of 8 ul with RT buffer (final concentration 1X), oligodT RT primer with the Illumina Read2 sequence (final concentration of 1 uM), and dNTP mix (NEB Cat# N0447L, final concentration 1 mM each). Samples were then transferred to a thermocycler at 95°C for 2.5 minutes, allowing for Mg2+-mediated RNA fragmentation, and then samples were fast cooled on ice. 0.5 ul of 4X RT buffer, 0.5 ul of 75 uM template switching oligo (TSO, which contained a sample index, UMI, variable length stagger region, and the Illumina Read1 primer sequence), and 1 ul of RT enzyme were added to each sample, and reverse transcription was performed according to manufacturer protocol. Samples with unique TSO-added sample indices were then pooled and treated with RNaseA (NEB Cat# T3018L, 1 ul per pooled sample) for 30 minutes at room temperature, and then cDNA was column purified (Zymo Cat# D4013) using 7 volumes of DNA binding buffer, and eluted in 36 ul of Buffer EB (Qiagen Cat# 19086). 3 ul of each reaction were amplified in a 1/10th scale reaction, as described below, with the addition of 1X EvaGreen (VWR Cat# 76423-472) in a QuantStudio 12k Flex (ThermoFisher Scientific), and the cycle number necessary to reach ~1/3 the maximum amplification was chosen for final pool amplification. The remaining ~30 ul of cDNA was amplified using the NEBNext Ultra™ II Q5 Master Mix (NEB Cat# M0544L) and NEBNext Multiplex Oligos for Illumina (NEB Cat# E7335S) according to manufacturer instructions and using the cycle number determined above. Final sequencing libraries were purified using SPRIselect beads (Beckman Coulter Cat# B23318), with a double-sided size selection (0.5X-0.8X). Libraries were sequenced on a NextSeq 2000 (P3 flowcell kit, 200 cycles, Illumina Cat# 20040560), with a 144 cycle Read1 which contained the sample index, UMI, and cDNA insert.   RNA-lite primers:  RNA-lite_RT-primer: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT [T-30]VN  RNA-lite_TSO: ACACTCTTTCCCTACACGACGCTCTTCCGATCT[Sample Index-8][UMI-8][Stagger-0-8 bases]­rGrGrG