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Sample GSM7360686

Query DataSets for GSM7360686

Status

Public on May 15, 2024

Title

HCT116-Chaetocin500nM-2-5H

Sample type

SRA

Source name

HCT116

Organism

Homo sapiens

Characteristics

cell line: HCT116
cell type: colorectal carcinoma
replicate: biol rep 2
treatment: Chaetocin, 500 nM
time: 5h

Treatment protocol

Cells were treated with solvent control (DMSO, 0.5%), chaetocin, chetomin or their respective sulfide-depleted analogs to final concentration of 500 nM for 5 H or 12 H.

Growth protocol

HCT116 cells were seeded in 6-well plates at 2 × 10^5 cells/well in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Sigma) and 1% Antibiotic-Antimycotic solution (Gibco), left overnight to attach and acclimate.

Extracted molecule

total RNA

Extraction 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).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

diff_expr_genes_all_cpm1_5H.xlsx

Data processing

Reads were mapped to the human transcriptome (GRCh38) with STAR, version 2.5.2b.
Gene expression was quantified using RSEM, version 1.3.3.
Differential gene expression analysis was performed with the edgeR package.
Assembly: GRCh38
Supplementary files format and content: Fold changes, P-values, and normalized counts for every gene in each sample.

Submission date

May 16, 2023

Last update date

May 15, 2024

Contact name

Alberto Riva

E-mail(s)

ariva@ufl.edu

Organization name

University of Florida

Department

ICBR