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Sample GSM7021875

Query DataSets for GSM7021875

Status

Public on Oct 03, 2023

Title

GN015 EV

Sample type

SRA

Source name

Colon

Organism

Homo sapiens

Characteristics

tissue: Colon
cell type: FACS sorted Live+singlets+CD3+TCRgd+Vd2neg-Vg234+
treatment: Co-culture over 16hrs with 293T cells expressing EV

Treatment protocol

5x105 HEK293T cells, transduced with either empty vector (EV) or BTNL3+8 (transduction is described in Di Marco Barros et al Cell 2016; 167: 203-218) and 2x105 freshly harvested primary human lymphocytes were co-cultured in 96-well plates with complete medium without supplementary cytokine and incubated at 37C at 5% CO2 for 16hrs

Growth protocol

Primary gut lymphocytes were obtained using an adaptation of the method of Kupper and Clarke (Clark et al., 2006) 9mm x 9mm x 1.5mm Cellfoam matrices (Cytomatrix PTY Ltd), were autoclaved and incubated in 100mg/mL rat tail collagen I (BD Biosciences) in PBS for 30min at 37 C, and washed twice in PBS. In compliance with local ethical approval, 12 endoscopic biopsies were taken from the ascending colon of donors. Biopsies were washed for 20min in 5mL wash medium (RPMI 1640 10% FCS, b-mercaptoethanol, penicillin [500U/ml], streptomycin [500 mg/ml], metronidazole [5 mg/ml, Pharmacy department, Guy’s Hospital], gentamicin [100 mg/ml, Sigma-Aldrich] and amphotericin 12.5 mg/ml [Thermo Fisher Scientific]). One endoscopic biopsy was placed on top of each matrix, which was inverted, and pressure applied, to crush the biopsy into the matrix. The matrices were placed into a 24-well plate (1 per well) and covered with 2mL RPMI 1640 (supplemented with 10% FCS, b-mercaptoethanol, penicillin [100U/ml], streptomycin [100 mg/ml], metronidazole [1 mg/ml], gentamicin [20 mg/ml], amphotericin [2.5 mg/ml]), IL-2 (100U/mL, Novartis Pharmaceutical UK) and IL-15 (10ng/mL, Biolegend). 1ml of medium was aspirated every second day and replaced with complete medium containing 2x concentrated cytokines. Cells were harvested and residual biopsy and empty wells were washed with PBS 0.02mM HEPES. The cell suspension was passed through a 70 mm nylon cell strainer, centrifuged at 400 g for 5min and resuspended in complete medium without additional cytokine and placed into co-culture immediately. Lymphocytes were used after 5-7 days of culture.

Extracted molecule

total RNA

Extraction protocol

After overnight co-culture cells were labelled sorted and RNA was extracted using the RNeasy Micro Plus Kit (Qiagen)
cDNA was prepared with Ovation RNA-Seq System V2 (NuGEN) and libraries were constructed with Ultralow Library System V2 (NuGEN). Resulting libraries were pooled and sequenced on an Illumina HiSeq 2500 platform with single ended 75 bp reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

count_matrix.txt

Data processing

bcl2fastq2 v2.2.0
reads trimmed with cutadapt
aligned and quantified with STAR and RSEM
Assembly: GRCh38
Supplementary files format and content: tab delimited RSEM gene-level count matrix

Submission date

Feb 02, 2023

Last update date

Oct 03, 2023

Contact name

Philip East

Organization name

The Francis Crick Institute

Lab

Bioinformatics & Biostatistics

Street address

Midland Road