title: GSM9166001: NGS4790 BP043202 NCI-H2731 G03498025-1uM; Homo sapiens; RNA-Seq
library_construction_protocol: For treatment, cells were seeded at 1-2 × 10^5 cells/well on tissue culture treated 6-well plates, and were treated after 12-24 h with experimental compounds. Cell lines were lysed on-plate for RNA isolation using the Qiagen RNeasy Plus Mini Kit (catalog number 74034), and RNA concentration was determined using a NanoDrop 8000 (Thermo Fisher Scientific). For analysis by RNA Fluidigm, 100 ng RNA was subjected to a complementary DNA (cDNA) synthesis reaction using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Cat. No. 4368814), as per the manufacturer's protocol. A preamplification reaction was then performed with Taqman PreAmp Master Mix (Thermo Fisher Scientific). After amplification, samples were diluted 1:4 with Tris-EDTA, and qPCR was conducted on Fluidigm 96. RNA integrity was confirmed using a Bioanalyzer 2100 (Agilent Technologies). About 500 ng of RNA was used for library synthesis using the TrueSeq RNA Sample Preparation kit v2 (Illumina). Size of the libraries was confirmed using 2200 TapeStation and High Sensitivity D1K screen tape (Agilent Technologies), and the concentration was determined by a qPCR-based method using the Library Quantification Kit (KAPA). The libraries were multiplexed and then sequenced on Illumina HiSeq2500 (Illumina) to generate 30M single-end 50-base pair reads.