GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3514562

Query DataSets for GSM3514562

Status

Public on Apr 15, 2019

Title

HEK_a12eCBS_dCas9-VPR_sorted_HiC_rep2

Sample type

SRA

Source name

HEK293T cells

Organism

Homo sapiens

Characteristics

cell type: HEK293T cells
genotype: HEK293T cells positively transfected with dCas9-VPR targeting the eCBS of Pcdha12
chip antibody: N/A

Growth protocol

HEK293T cells were purchased from ATCC and cultured in DMEM supplemented with 10% (vol/vol) FBS, 1X GlutaMax, 1mM sodium pyruvate, 1X non-essential amino acids, 1% penicillin-streptomycin. Cells were maintained at 37°C in a 5% (vol/vol) CO2 incubator.

Extracted molecule

genomic DNA

Extraction protocol

Mice were sacrificed using CO2 followed by cervical dislocation. The main olfactory epithelium (MOE) was dissected and transferred to ice-cold PBS. The MOE was cut in to small pieces with a razor blade, and then dissociated with a papain dissociation system (Worthington Biochemical). For RNA-seq, dissociated cells were washed once with sort media (PBS + 2% Fetal Bovine Serum) and then resuspended in sort media supplemented with 100 U/mL DNase I (Worthington Biochemical), 4mM MgCl2, and 500ng/mL DAPI (Invitrogen). For Crosslinked-ChIP samples, cells were fixed prior to sorting. For fixation, dissociated cells were resuspended in PBS + 1% methanol-free formaldehyde (Pierce). Cells were fixed at room temperature for 5 minutes, and then fixation was quenched by adding 1/10th volume of 1.25M glycine. Fixed cells were pelleted (500 rcf, 5 minutes, room temperature), washed once with PBS + 2% FBS, and then resuspended in sort media. All cells were passed through a 40uM cell strainer prior to sorting.

Library strategy

Hi-C

Library source

genomic

Library selection

other

Instrument model

NextSeq 550

Description

captureHiC-Seq, Paired End
Processed data file includes data for rep1, 2 and 3. File is available on GSM3514561

Data processing

Adapter Sequences were trimmed with cutadapt (v1.9.1) (--minimum-length 18)
Alignment: All mouse samples were aligned to mm10, human samples aligned to hg19. For paired-end data sets, only read 1 was used for alignment and downstream analysis. Crosslinked ChIP-Seq samples were aligned with Bowtie2 (2.3.2) with default parameters. RNA-seq samples were aligned with STAR (v2.5.3a) with the following parameters: --outFilterIntronMotifs RemoveNoncanonical --outFilterType BySJout --outFilterMultimapNmax 20
Filtering: Unmapped reads and reads with a mapping quality below 30 were removed with Samtools (1.3.1). For ChIP-seq, duplicate reads were marked with Picard and removed with samtools.
For ChIP-Seq, bigWig files were generated using HOMER (4.7) (-fsize 1e20 -fragLength given). For RNA-Seq, bigWig files were generated with RSeQC (2.6.4).
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files at base pair resolution normalized to a library size of 10,000,000 reads

Submission date

Dec 14, 2018

Last update date

Apr 15, 2019

Contact name

Daniele Canzio

Organization name

University of California San Francisco

Department

Neurology

Street address

555 Mission Bay Boulevard South