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Sample GSM3687056

Query DataSets for GSM3687056

Status

Public on Aug 22, 2019

Title

SUM159_shEN1-2_NoDoxy_2L

Sample type

SRA

Source name

SUM159PT

Organism

Homo sapiens

Characteristics

hairpin: shEN1-2
treatment: No Dox

Treatment protocol

Xenograft RNA-seq: When the tumors became palpable, shRNAs were induced in the treatment group by administering a doxycycline diet (625ppm). Animals were euthanized and tumors were harvested when tumors in the control group reached ~ 1.5 cm size

Growth protocol

Xenograft RNA-seq: Animal experiments were performed following protocol 11-023 approved by DFCI ACUC. SUM149 or SUM159 cells (1 x 106) expressing TET-inducible Luciferase or EN1-targeting shRNAs were resuspended in 50% Matrigel (BD Biosciences) and injected orthotopically into the mammary fat pads of 6-week old female NOG mice (Taconic).

Extracted molecule

total RNA

Extraction protocol

ChIP-seq: cell monolayers were fixed with 1% formaldehyde for 10 min, at room temperature for H3K27ac and FOXA1 ChIP-seq, or 37C for V5-EN1 and TLE3 ChIP-seq. Cells were fixet with 1.5mM EGS during 30 min and adding 1% paraformaldehyde during the last 10 min for b-catenin ChIP-seq. All cell fixations were wuenched by adding 125mM glycine during 5 min. After 2 ice-cold PBS washes, cells were scrapped off in PBS, pelleted and snap-frozen. RNA-seq: cells were washed 3 times with ice-cold PBS, scrapped off the plates, pelleted and snap frozen into liquid nitrogen. For RNA-extraction, the RNeasy Mini Kit (Qiagen, #74106) was used, with in-column DNA digestion.
ChIP-seq: the ThruPLEX DNA-seq Kit (Rubicon Genomics, R400427) was used following manufacturer's instructions. RNA-seq: libraries were prepared using Illumina TruSeq Stranded mRNA sample preparation kits from 500ng of purified total RNA according to the manufacturer’s protocol

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

20190207_SUM159_shEN1-2_NoDoxy_2L_GP6615_S6
RNA-seq of SUM159 PDX cells with TET-inducible shEN1-2 without doxycycline
SUM159_shEN1-2_NoDoxy_2L.Aligned.sortedByCoord.out.hg19.mm10-filtered

Data processing

ChIP-Seq: QC, alignment and peak calling was performed using ChiLin pipeline 2.0.0 (https://www.ncbi.nlm.nih.gov/m/pubmed/27716038/?i=6&from=chilin). Briefly, reads were aligned to GRCh37/hg19 using BWA, and peak calling was performed using MACS2, in chilin.
RNA-Seq: Datasets were aligned to the human reference GRCh37/hg19 genome and to the mouse reference GRCm38/mm10 genome, both using the STAR RNA-Seq aligner (version STAR_2.5.1b). Two-pass mapping was performed using the following modified parameters: --outSAMstrandField intronMotif, --outFilterMultimapNmax 20, --alignSJoverhangMin 8, --alignSJDBoverhangMin 1, --outFilterMismatchNmax 999, --outFilterMismatchNoverLmax 0.1, --alignIntronMin 20, --alignIntronMax 1000000, --alignMatesGapMax 1000000, --outFilterType BySJout, --outFilterScoreMinOverLread 0.33, --outFilterMatchNminOverLread 0.33, --limitSjdbInsertNsj 1200000, --chimSegmentMin 15, --chimJunctionOverhangMin 15, --twopassMode Basic. Samples were filtered to remove contamination (mouse contamination). Reads uniquely mapped only to the target genome were kept, along with uniquely mapped reads that had significantly better alignment scores in the target genome compared to the contamination genome.
Supplementary_files_format_and_content: RNA-Seq: Tab-separated file of the raw read counts of each samples for each gene.
Supplementary_files_format_and_content: ChIP-Seq: bed file (BED6+4) of factor peaks from MACS2 peakcaller.

Submission date

Mar 25, 2019

Last update date

Aug 22, 2019

Contact name

Kornelia Polyak

E-mail(s)

kornelia_polyak@dfci.harvard.edu

Phone

617-632-2106

Organization name

Dana-Farber Cancer Institute