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Sample GSM5729389

Query DataSets for GSM5729389

Status

Public on Aug 08, 2022

Title

IL9: IL9 treated colonoid donor 4

Sample type

SRA

Source name

colonic organoid

Organism

Homo sapiens

Characteristics

disease state: healthy
tissue: colonic organoid
treatment: IL9 10ng/mL
treatment duration: 24 hours
donor: 4
experiment: 2

Treatment protocol

During the last 24h in differentiation medium human colonoids were treated with human recombinant IL13 (10 ng/mL) or IL9 (10ng/mL).

Growth protocol

Establishment of human colonoids was performed as previously described by Fujii et al.(2015). The crypts were cultured in growth medium containing advanced Dulbecco’s modified Eagle’s medium/F12, penicillin/streptomycin (100 units/mL), 10 mM HEPES, 2 mM Glutamax, supplements N2 (1x) and B27 (1x), 50 ng/mL mouse epidermal growth factor (all from Life Gibco), 1 mM N-acetylcysteine (Sigma-Aldrich), 50% v/v Wnt3a conditioned medium, 10% v/v R-spondin-1 conditioned medium, 100 ng/mL murine recombinant noggin protein (Peprotech),10 nM gastrin (Sigma-Aldrich), 500 nM A83-01 (Bio-techne), 10 μM SB202190 (Sigma-Aldrich) and 10 mM Nicotinamide (Sigma-Aldrich). 10 μM Y-27632 (Sigma-Aldrich) was added to the culture medium for the initial 3 days. Medium was changed every 2 days. Differentiation towards a mature epithelium in human colonoids was achieved with reduction of Wnt3a to 15% v/v and withdraw of SB202190 and nicotinamide for 5-7 days.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from human colonoids (released from Matrigel with Cell Recovery solution (Corning)) using RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. On-column DNase digestion was performed to remove any residual genomic DNA (Qiagen).
mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptors with hairpin loop structure were ligated. The library fragments were then purified with AMPure XP system (Beckman Coulter, Beverly, USA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Fastq files were firstly processed with in-house Perl scripts to discard reads with adaptor contamination, or at least 10% of uncertain bases (N), or at least 50% of nucleotides with a Phred quality score less than 20
Read pairs were then aligned to the human genome (GRCh37/hg19) using TopHat v2.0.12
HTSeq v0.6.1 was used to count the read pairs mapped uniquely and concordantly to each gene
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: files in tab-separated value format report the raw counts of read pairs uniquely and concordantly mapped to each gene

Submission date

Dec 11, 2021

Last update date

Aug 08, 2022

Contact name

Nick Powell

E-mail(s)

npowell@ic.ac.uk

Organization name

Imperial College London

Department

Department of Metabolism, Digestion and Reproduction