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Sample GSM3530286

Query DataSets for GSM3530286

Status

Public on Dec 26, 2018

Title

FANTOM10-39721055

Sample type

SRA

Source name

Colonic macrophages

Organism

Homo sapiens

Characteristics

cell type: CD45+ CD64+ HLA-DR+ CD206+

Treatment protocol

Intestinal tissue processing. To remove the epithelial layer from biopsies, tissue was incubated in 2 mM EDTA/HBSS (Gibco) for three minutes at 37°C in a shaking incubator. For resected tissue samples, fat and muscle layers were first removed; tissue was washed in HBSS and subsequently dissected into 0.5 cm pieces. To remove the epithelial layer from resections, tissue was incubated in 2 mM EDTA/HBSS for 15 minutes at 37°C in a shaking incubator. Tissue was vigorously shaken; supernatant discarded and the process was repeated three times. After the final EDTA step, all samples were washed in HBSS and resuspended in 2 ml (20 ml for resections) of pre-warmed 10% complete media (RPMI 1640 supplemented with 10% foetal calf serum (FCS), 100 U ml-1 penicillin, 100 U ml-1 streptomycin, 2 mM L-glutamine and 50 μM 2-mercaptoethanol) containing: Collagenase VIII (1 mg/ml, Sigma-Aldrich), Collagenase D (1.25 mg/ml, Roche), Dispase (1 mg/ml, Gibco) and DNase. Samples were returned to the shaking incubator (37°C) for 30-40 minutes; additional vigorous shaking was performed every 10 minutes to aid digestion. Cell suspension was first filtered through a 100 μM then a 40 μM cell strainer (BD Falcon) and washed in PBS containing 2 mM EDTA and 2% FCS.

Extracted molecule

total RNA

Extraction protocol

Sample preparation: Histologically normal margins of healthy colonic resections were processed, digested and stained for FACS analysis as previously described. CD45+ CD64+ HLA-DR+ CD206+ and CD45+ CD64+ HLA-DR+ CD206- cells were sorted using a FACSAria II/III. Cells were sorted into 300 μl RLT buffer (Qiagen) and stored at -80°C prior to RNA extraction using a MicroRNA kit (Qiagen).
Sequencing libraries were prepared using the SMARTer Stranded Total RNA-Seq kit-Pico Input Mammalian (Clontech), starting with 1ng of RNA and following the manufactures protocol with 15 cycles of PCR.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

FANTOM_10

Data processing

the sequencing was done on the Illumina NextSeq 500 using High Throughput flow-cells multiplexing eight samples per flow cell to produce 50M read-pairs per sample on average.
Sample-specific fastq files were generated using bcl2fastq software.
Statistical analysis: Data was aligned using HISAT2 and the reads summarised and normalised using the Feature Counts tool.
Differential expression was performed using the DESeq2 R package. Significant differential expression was defined as adjusted P-value < 0.05.
Supplementary_files_format_and_content: Deseq files contain abundance table comparisons

Submission date

Dec 25, 2018

Last update date

Dec 27, 2018

Contact name

Simon Milling

E-mail(s)

Simon.Milling@glasgow.ac.uk

Phone

+44 1413306419

Organization name

University of Glasgow