 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 21, 2019 |
| Title |
TTC1240_ChIP_N106_SMARCB1_K364del_DPF2 |
| Sample type |
SRA |
| |
|
| Source name |
tumor cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: TTC1240
ngs approach: ChIP-seq
experimental condition: lentivirus: SMARCB1 K364del
chip antibody: DPF2/BAF45D (Abcam, ab128149, lot: GR250670-5)
|
| Treatment protocol |
Lentiviral particles were prepared in 293T packaging cells via polyethylenimine-mediated transfection as previously described (Bell et al 2012). TTC1240 and G401 cells were lentivirally infected with either Empty vector, or one of four SMARCB1 variant constructs (full length, K364del, R377H, or delCC construct) for 48h and then selected with blasticidin for 5 days. Cells were harvested 7 days post-infection. Treatment: The SAH iPSC cell line underwent CRISPR/Cas9 mediated genome editing with a short-guide RNA (sgRNA) targeting exon 9 of the SMARCB1 gene and a single-stranded oligodeoxynucleotide (ssODN) donor strand encoding for the SMARCB1 K364del in-frame deletion. Cells were single cell sorted, genotyped and then confirmed via standard TA-cloning procedures to onbtain a SMARCB1+/+ WT control and K364del hetorzygous mutant (SMARCB1^K364del/+). The SMARCB1^K364del/+ iPSC cells were made into stable lines via lentiviral infection with either Empty vector or full-length SMARCB1 followed by blasticidin selection. Ngn2 differentiation of SAH cells: At Day (-2) cells were split and plated at 2K/well in geltrex coated plates. To induce NGN2-mediated differentiation, cells were infected with NGN2 lentiviral construct conjugated with EGFP and rTTA cassette on Day (-1). Media was changed periodically according to standard protocol.
|
| Growth protocol |
TTC1240 and G401 cells were grown in DMEM (Gibco, 11960-44), supplemented with 10% FBS, 1X GlutaMAX (Gibco, 35050-061), 100 U/mL penicillin-streptomycin (Gibco, 15140-122), 1mM sodium pyruvate (Gibco, 11360-070), 1X MEM NEAA (Gibco, 11140-050), 10 mM HEPES, 1X 2-mercaptoethanol. The SMARCB1^K364del/+ iPSC cells were maintained in StemFlex Medium (Gibco) prior to differentiation. At Day (-2) of NGN2-mediated differentiation, the iPSCs were washed with 1X PBS (Gibco), and dissociated using Accutase (Innovative Cell Technologies).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq was performed using standard protocols (Millipore, Billerica, MA). Specifically, cells were fixed in 1% formaldehyde (Sigma Aldrich, F8775) for 10min at 37C and quenched with 125mM glycine for 5min at 37C. After washing, nuclei were sonicated using Covaris Sonicator, and the supernatant was used for immunoprecipitation with the indicated antibody. All ChIP-seq was sequenced on Illumina Next-seq 500 using 75 bp single-end sequencing parameters. RNA was collected in biological duplicate using the RNeasy Mini Kit (QIAGEN). All RNA was sequenced on Illumina Next-seq 500 using 75 bp single-end sequencing parameters. ATAC-seq libraries were prepared in biological replicates using standard protocol (Buenrostro et al., Current Protocols (2013)) with 12 cycles of amplification. ATAC-seq samples were sequenced on Nex-seq 500 using 37 bp, 37 bp pair-end sequencing parameters.
ChIP-sequencing libraries were prepared with Illumina’s NEBNext Ultra II DNA library Prep Kit using standard protocols. RNA-seq libraries were prepared with Illumina’s TruSeq standard mRNA Sample Prep Kit using standard protocols. ATAC-seq libraries were prepared using a standard protocol (Buenrostro et al., Current Protocols (2013)).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Illumina NextSeq output data were demultiplexed and converted to FASTQ format using the bcl2fastq software tool.
RNAseq reads were aligned to the hg19 genome with STAR v2.5.2b, and ChIPseq reads were aligned with Bowtie2 v2.29 in the -k 1 reporting mode.
For the ATACseq and Mnase-seq data, quality read trimming was performed by Trimmmomatic v0.36, followed by alignment, duplicate read removal, and read quality filtering, using Bowtie2 v2.29, Picard v2.8.0, and SAMtools v0.1.19, respectively.
For the ChIPseq, ATACseq and Mnase-seq data, output BAM files were converted into BigWig track files using BEDTools and UCSC utilities in order to display coverage throughout the genome (in RPM).
For the RNAseq data, tracks were generated using the deepTools v2.5.3 bamCoverage function.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig files give the normalized coverage of RNA or DNA fragments across the genome in reads per million.
|
| |
|
| Submission date |
Jan 25, 2019 |
| Last update date |
Nov 21, 2019 |
| Contact name |
Cigall Kadoch |
| E-mail(s) |
cigall_kadoch@dfci.harvard.edu
|
| Organization name |
Broad Institute of MIT and Harvard, Harvard Medical School, Dana-Farber Cancer Institute
|
| Street address |
450 Brookline Avenue
|
| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE124903 |
mSWI/SNF functional genomic characterization of SMARCB1 mutants in SMARCB1-null and heterozygous settings |
|
| Relations |
| BioSample |
SAMN10822031 |
| SRA |
SRX5294340 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3580470_TTC1240_ChIP_N106_SMARCB1_K364del_DPF2.bw |
172.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
