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Sample GSM1921006

Query DataSets for GSM1921006

Status

Public on Nov 12, 2015

Title

BAFi_rep1_RNAseq

Sample type

SRA

Source name

human keratinoctyes with Brg1/Brm siRNA

Organism

Homo sapiens

Characteristics

cell type: primary human neonatal keratinocytes
genotype/variation: Brg1/Brm knockdown

Treatment protocol

To induce differentiaiton, keratinocytes were seeded in confluence and cultured with the addition of 1.2mM of calcium to growth medium. 1nM of siRNA for each target were nucleofected (Lonza to keratinocytes)

Growth protocol

neonatal keratinocyte were isolated from discarded foreskin, cultured in KSF and 154 keratinocyte medium

Extracted molecule

total RNA

Extraction protocol

For ATAC-seq, around 50,000 cells were used for each transposition reaction. Nuclei were prepared prior to transposition. For ChIP-seq, a mininum of 2 million cells were crosslinked with 1% formaldehyde, sonicated, and immunoprecipiated with antibodies recognizing Brg1/Brm (J1), H3K27Ac, H3K27me3, H3K4me1, p300, pol II, and p63. For RNA-seq, RNA were extracted using RNeasy Plus (Qiagen), rRNA were removed using using Ribo-Zero rRNA Removal Kit (Illumina).
For ATAC-seq, sequencing libraries were constructed using a modified version of the Illumina Nextera DNA Sample prep kit. For ChIP-seq, NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina was used for library construction. For RNA-seq, NEBNext Ultra RNA Library Prep Kit was used for library construction.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Reads were trimmed for adaptor sequence, then mapped to UCSC hg19 using bowtie, duplicate fragments were then removed using Picard.ATACseq peaks were called using the MACS2 algorithm,ChIPseq peaks were called using MACS14.
Number of Raw reads in each peak (or each gene for RNAseq) was calculated using in house generated script, and data matrix was normalized using R. For RNAseq, data normalization of significant analysis was performend using Deseq.
Genome_build: hg19
Supplementary_files_format_and_content: normalized read counts for each gene (RNA-seq) is in a text file.

Submission date

Oct 28, 2015

Last update date

Oct 11, 2022

Contact name

Douglas Porter

Organization name

Stanford

Department

Dermatology

Lab

Khavari