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Sample GSM3039693

Query DataSets for GSM3039693

Status

Public on Jan 01, 2019

Title

GFP-transduced [KLF4WT] Bio rep.3 Tech rep c

Sample type

SRA

Source name

skin, epidermis

Organism

Homo sapiens

Characteristics

cell type: holoclone cells, keratinocyte precursor cells
biological samples: adult skin biopsies (breast surgery)
culture history: 55 population doublings post-initial clonal isolation

Treatment protocol

Lentiviral vectors were used for stable gene knock-down in holoclone cells (keratinocyte precursor cells). Transduction was performed on cultured cells at 50 population doublings after initial clonal derivation and at 20 % confluence. Cells were incubated overnight with lentiviral particles in the presence of hexadimethrine bromide. After 3 days, keratinocytes at 80 % confluence were collected and analyzed by flow cytometry. Transduced cells were sorted according to their GFP fluorescence.

Growth protocol

Human skin tissue from adult healthy donors was collected in the context of breast reduction surgery, after informed consent. Epidermal keratinocytes were extracted after enzymatic treatment. Frozen banked samples of human epidermal holoclone keratinocytes were studied as a model of skin keratinocyte precursor cells. Mass cultures of holoclone cells (keratinocyte precursor cells) were performed in a serum-containing medium, in the presence of a feeder-layer of human dermal fibroblasts growth-arrested by γ irradiation (60 Gy). Cultures were performed in plastic devices coated with type I collagen. Medium was renewed 3 times a week, and sub-cultured every week. Numbers of population doublings achieved by cultures were calculated after each passage, as follows: PD = (log N/N0)/log 2, where N0 represents the number of plated cells, and N the number of cells obtained after 1 week of growth.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA from these 9 samples was extracted with RNeasy Mini and Micro kits (Qiagen) followed by quality control using capillary electrophoresis (RNA 6000 Nano chips, Agilent).
Libraries were prepared according to Illumina's instructions with theTruSeq RNA Library Prep Kit v2. Briefly, total RNA (1µg) was purified and fragmented before first strand and second strand cDNA synthesis. Then ends are repaired and 3’ends adenylated before adapters ligation and PCR amplification. Libraries were then validated, normalized, and sequenced on Illumina HiSeq 1000 as short-insert paired-end libraries with read lengths 100 bp.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 1000

Data processing

Base Calling: Basecalls performed using CASAVA version 1.4
Read Quality: Quality of raw reads was assessed using FastQC quality control version 0.11.3 http://www.bioinformatics.babraham.ac.uk/projects/fastqc
Mapping: Primary assembly Homo sapiens genome sequence (release GRCh38.p3) and transcriptome annotations (Ensembl release 81) were downloaded from the GENCODE website2 (files GRCh38.primary_assembly.genome.fa and gencode.v23.primary_assembly.annotation.gtf respectively). Raw read data (fastq files) were mapped to these sequences using STAR
Merging: For each biological replicate the 3 technical replicates were merged
Normalization: For the quality control representations, the effective library sizes were first computed using function estimateSizeFactors from package DESeq24. The raw count values were then transformed using a regularized log (function rlogTransformation from package DESeq2 with parameter blind=TRUE).
Gene filtering: Genes that did not have more than one count per million counts in at least 2 samples were filtered out. Genes were also filtered if their Ensembl description was either empty, “Uncharacterized protein” or an open reading frame.
Genome_build: GRCh38
Supplementary_files_format_and_content: raw and normalized counts

Submission date

Mar 13, 2018

Last update date

Jan 01, 2019

Contact name

Nicolas Fortunel

E-mail(s)

[email protected]

Phone

+33160873492

Organization name

CEA