title: GSM7360703: HCT116-Chaetocin500nM-2-12H; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was extracted using RNeasy Mini Kit (Qiagen) according to manufacturer instructions. Illumina RNA library construction and sequencing were performed at the Interdisciplinary Center for Biotechnology Research (ICBR) Gene Expression Core, University of Florida (UF) ..  For library construction, RNA samples were measured by the QUBIT fluorescent method (Invitrogen) and Agilent Bioanalyzer. An amount of 250 ng of high quality of total RNA with RIN of 7 or higher was used for library construction using the reagents provided in the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, catalog # E7490) and the NEBNext Ultra II Directional RNA Library Prep Kit (New England  Biolabs, catalog #E7760) according to the manufacturer's user guide. Briefly, 200 ng of total RNA was used for mRNA isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, catalog # E7490). Then the poly A enriched RNA was fragmented in NEBNext First Strand Synthesis Buffer via incubation at 94 °C for the desired time. This step was followed by first-strand cDNA synthesis using reverse transcriptase and random hexamer primer.  Synthesis of ds cDNA is done using the 2nd strand master mix provided in the kit, followed by end-repair and dA-tailing.  At this point, Illumina adaptors are ligated to the sample.  Finally, library was amplified, and followed by purification with AMPure beads (Beckman Coulter, catalog # A63881).  The library size and mass were assessed by analysis in the Agilent TapeStation using a High Sensitivity DNA1000 Screen Tape.  Typically, a 250-900 library peak is observed with the highest peak at ~420 bp. Barcoded libraries were pooled equimolarly for sequencing simultaneously for NavaSeq 6000 S4 2x150 cycles run as described below aiming for 50 million reads per sample. RNA-seq library performed at UF ICBR Gene Expression Core (https://biotech.ufl.edu/gene-expression-genotyping/, RRID:SCR_019145).   For Illumina NovaSeq6000 sequencing, Normalized libraries were submitted to the “Free Adapter Blocking Reagent” protocol (FAB, Cat# 20024145) in order to minimize the presence of adaptor-dimers and index hopping rates.  The library pool was diluted to 0.8 nM and sequenced on one S4 flow cell lane (2x150 cycles) of the Illumina NovaSeq6000. The instrument's computer utilized the NovaSeq Control Software v1.6. Cluster and SBS consumables were v1.5. The final loading concentration of the library was 120 pM with 1% PhiX spike-in control. One lane generated 2.5-3 billion paired-end reads (~950Gb) with an average Q30%>= 92.5% and Cluster PF= 85.4%. FastQ files were generated using the BCL2fastQ function in the Illumina BaseSpace portal. The Illumina NovaSeq 6000 was used to sequence the libraries for 2 x 150 cycles. Sequencing was performed at the ICBR NextGen Sequencing (https://biotech.ufl.edu/next-gen-dna/, RRID:SCR_019152).