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Sample GSM2698819

Query DataSets for GSM2698819

Status

Public on Sep 03, 2018

Title

Huh6 1.33

Sample type

SRA

Source name

liver cancer Huh6 cells

Organism

Homo sapiens

Characteristics

genotype: STRAP KO

Growth protocol

Huh6 and Huh7 cell lines were transfected in 6-well plates using 7.5 µl FuGENE® HD Transfection Reagent (E2311, Promega) and 2 µg of each pX330 plasmid per well together with 0.2 µg GFP expression construct. GFP expression was used to select the cells that received high levels of the pX330 CRISPR/Cas9 constructs. After incubation at 37°C for 24 h, single cells were prepared for fluorescence activated cell sorting to a 96-well plate. After single cell sorting, Huh7 cells were maintained in DMEM supplemented with either 20% FCS or 25% Huh7-conditioned medium. Huh6 and PLC/PRF/5 were cultured in complete DMEM medium.

Extracted molecule

polyA RNA

Extraction protocol

RNA was isolated with the Machery-NucleoSpin RNA II kit (BIOKE, Leiden, The Netherlands)
RNA-seq was performed at GATC Biotech (Konstanz, Germany) according to manufacture's instructions (Illumina). Briefly, the mRNA was enriched using oligo-dT magnetic beads, followed by fragmentation (about 200 bp). Then the first strand of cDNA was synthesized using random hexamer-primer and the second strand was further synthesized in a reaction buffer including dNTPs, RNase H and DNA polymerase I. Double stranded cDNA was purified with magnetic beads. Then, the 3’-end single nucleotide A (adenine) was added and adapters were ligated to the fragments which were enriched by PCR amplification.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

RNA isolation date 9/18/2016

Data processing

The Illumina single-end reads were trimmed to remove the TrueSeq adapter sequences using Trimmomatic (v.0.33).
The reads were mapped to the human reference genome build hg38 with the RNA-seq aligner STAR (v2.4.2a) and the Homo sapiens GENCODE v23 annotation.
Raw counts were measured with summarizeOverlaps function from the Bioconductor GenomicAlignments package (v1.12.1) using the setting mode union.
The differentially expressed genes were called with a generalized linear model using a negative binomial distribution and accounting for the different cell lines (Huh6 and Huh7). The calculations were performed by the DESeq2 package (v1.16.1).
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files include Ensemble Gene Id and read count

Submission date

Jul 10, 2017

Last update date

May 15, 2019

Contact name

Wenhui Wang

E-mail(s)

w.wang.1@erasmusmc.nl

Organization name

Erasmus MC

Street address

Wytemaweg 80