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Sample GSM2934430

Query DataSets for GSM2934430

Status

Public on Jul 27, 2020

Title

mCherry - 2

Sample type

SRA

Source name

brown adipocyte

Organism

Homo sapiens

Characteristics

cell type: H1 hES derived
cell subtype: mCherry -

Growth protocol

UCP1::mCherry H1 hES cells were differentiated according to a published protocol (Ahfeldt et al., 2012). Briefly, reporter cells were placed into suspension culture to form embryoid bodies for 7 days, after which embryoid bodies were plated onto 0.1% gelatin coated dishes with DMEM supplemented with 10% FBS and 10 µg/mL bFGF to promote mesenchymal cell outgrowth. After reaching 90% confluency, cells were trypsinized and passaged for 4-5 rounds prior to plating for infection with doxycycline inducible PPARγ, CEBPβ, and PRDM16 lentiviral constructs. These cells were placed into adipogenic medium containing KOSR, dexamethasone, insulin, and rosiglitazone, along 700 ng/mL doxycycline for 14 days. Finally, doxycycline was removed and the cells were allowed to mature for another 6 days or longer as experiments required.

Extracted molecule

total RNA

Extraction protocol

For isolation of mCherry positive and negative cells for RNA-seq, differentiated cells were digested with 0.25% trypsin for 15 minutes until cells detached from plates. Cells were then digested with Liberase TM (Sigma, #5401119001) at 0.04 µg/mL in 2% BSA/DPBS on a rocking shaker at 37 degrees for 1 hour. Cell suspension was then filtered through a 70 μm mesh strainer and stained with DAPI prior to cell sorting. Cells were sorted on a BD Influx cell sorter equipped with 488 nm laser as the FSC/SSC source, a 355 nm laser for excitation of DAPI, and a 561 nm laser for excitation of mCherry. Cells were sorted directly into 750 µL lysis buffer using the Agilent Absolutely RNA Microprep kit (Cat#400805).
cDNA libraries were generated using the Illumina TruSeq stranded mRNA Sample Preparation kit and sequenced on HiSeq4000 sequencer.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

mCherry_negative_2

Data processing

Illumina bcl2fastq2 v2.19 was used for basecalling.
Sequenced reads were trimmed for low-quality bases and for adapter sequences using cutadapt; then mapped to the human hg19 reference genome using STAR v2.5.2b.
Raw reads counts were extracted using htseq-count v0.6.1.
Differential expression analysis was performed by using DESeq2 package v1.6.3.
Genome_build: hg19
Supplementary_files_format_and_content: excel file including gene name, Ensembl gene id, normalized read counts (log scale) for each gene in each sample

Submission date

Jan 12, 2018

Last update date

Jul 27, 2020

Contact name

Shuibing Chen

E-mail(s)

shuibing.chen@gmail.com

Phone

2127465431

Organization name

Weill Cornell Medical College