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Sample GSM7141043 Query DataSets for GSM7141043
Status Public on Jun 08, 2023
Title InputB_sgKDM6A_236R_1
Sample type SRA
 
Source name sgKdm6a RPP 236R
Organism Mus musculus
Characteristics cell line: sgKdm6a RPP 236R
cell type: mouse small cell lung cancer cell line (SCLC) derived from a mouse tumor
treatment: None
Treatment protocol No treatment
Growth protocol Early passage 236L and 236R cell lines derived from sgKdm6a RPP tumors (Kdm6a-Mutant) and early passage 1014 and 159-1 cell lines derived from sgControl RPP tumors (Kdm6a-WT) grown in complete media containing [10% FBS, P/S, and HITES (10 nM hydrocortisone (Sigma Aldrich # H0135), Insulin-Transferrin-Selenium (Gemini #400-145), and 10 nM beta-estradiol (Sigma Aldrich# E2257), 100 U/mL of penicillin (P), and 100 µg/mL of streptomycin (S)], were plated in ultra-low attachments plates at 0.4 X 10^6 cells/mL in 12mLs (4.8 X 10^6 cells total) or 20mLs (8 X 10^6 cells total) of complete media for H3K27me3/H3K4me2 or KDM6A ChIP, respectively. sgControl RPP cell lines (1014 and 159-1) were also plated at the same density to perform control IgG ChIP for each experiment. 24 hours later, cells were collected, and centrifuged. for ChIP experiments.
Extracted molecule genomic DNA
Extraction protocol KDM6A ChIP sequencing experiments were performed by first cross-linking cells with 2 mM disuccinimidyl glutarate (DSG) with rocking for 30 minutes at room temperature prior to fixation with 1% paraformaldehyde diluted in PBS for 10 minutes at room temperature. H3K4me2 and H3K27me3 ChIP was performed by fixation with 1% paraformaldehyde diluted in PBS for 10 minutes at room temperature. Excess formaldehyde was quenched by dropwise addition of freshly made Glycine at a final concentration 1.25 M. The cells were then resuspended in 1 mL of lysis buffer (50mM Hepes-NaOH pH 8, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% TX-100 supplemented with a protease inhibitors and phosphatase inhibitors) and incubated by rotating for 10 minutes at 4°C. Intact nuclei were then collected by centrifugation at 1900 x g for 5 minutes at 4°C. The supernatant was then gently aspirated and the pellet was resuspended in 1 mL of wash buffer (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, supplemented with a protease inhibitor cocktail and phosphatase inhibitors) and incubated by rotating for 10 minutes at 4°C. The nuclei were again collected by centrifugation at 1900 x g for 5 minutes at 4°C. The supernatant was then gently aspirated and the tube was rinsed with 500 µL of shearing buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris-HCl pH 8.0 supplemented with protease inhibitors and phosphatase inhibitors). The nuclei were again collected by centrifugation at 1900 x g for 5 minutes at 4°C, the supernatant was carefully removed, and the pellet was gently resuspended in 1mL shearing buffer and transferred to a 1mL AFA fiber tube (Covaris #520130). The chromatin was then sonicated using a Covaris E220 sonicator at 100 peak incident power, 10% duty cycles, 200 cycles per burst, water level 15 (left indicator)/10 (right indicator) for 900 seconds. The samples were then transferred to 1.5 mL Eppendorf tubes and centrifuged in an Eppendorf microcentrifuge at 15,900 x g for 5 minutes at 4°C. 900 µL of the supernatants were transferred to a new Eppendorf tube, to which 1% Triton X-100 and 150 mM NaCl were added. 20µl of Dynabeads Protein G (ThermoFisher #10004D), prewashed with Shearing Buffer, was then added and samples were rotated for 1 hour at 4°C to pre-clear the sonicated chromatin. The Dynabeads were then removed using a magnetic stand and the supernatants were transferred to new tubes. 2.5% of the total lysate was removed to make genomic DNA to be used for the ChIP-sequencing input DNA. For the H3K27me3 and H3K4me2 ChIP, the remaining lysate (95% of total) were equally distributed into two Eppendorf tubes to perform the H3K27me3 and H3K4me2 ChIPs. 3 µg of the rabbit anti-H3k27me3 (C36B11) (Cell signaling, #9733) or 3 µg of the rabbit anti-H3K4me2 (Abcam #Ab32356) primary antibodies were added. For the KDM6A ChIP, the remaining 95% were incubated with 10 µg of the rabbit anti-KDM6A [Bethyl lab, A302-374A (UTX antibody)]. Same amount of rabbit IgG isotype control (Cell Signaling #3900) with the same amount of lysate was used as a control for each ChIP. All samples were incubated overnight while rotating at 4°C. The following morning, 50µL of Dynabeads, prewashed with Shearing buffer, were added and the samples were incubated for another 2 hours while rotating at 4°C. A magnetic stand was used to isolate the Dynabeads. The supernatant was removed and the Dynabeads were washed once in 1mL of low salt immune complex wash buffer (20mM Tris-HCl pH 8.0, 150mM NaCl, 0.1% SDS, 1% TX-100, 2mM EDTA) and incubated for 5 minutes while rotating at 4°C. The beads were again isolated using a magnetic stand. Following removal of the supernatant the Dynabeads were washed once in 1mL of high salt immune complex wash buffer (20mM Tris-HCl pH 8.0, 500mM NaCl, 0.1% SDS, 1% TX-100, 2mM EDTA) and incubated for 5 minutes while rotating at 4°C. The beads were again pelleted with a magnetic stand. Following removal of the supernatant, the Dynabeads were washed once in 1mL of Lithium Chloride immune complex wash buffer (10mM Tris-HCl pH 8.0, 250mM LiCl, 1% SDS, 1% NP-40, 1mM EDTA) and incubated for 5 minutes while rotating at 4°C. The beads were then washed twice in TE pH8 at room temperature and the beads were then resuspended in 100µL TE pH8.
RNase A (Qiagen) was then added to a final concentration of 0.2 mg/mL and the samples were incubated for 30 minutes at 37°C. To reverse the formaldehyde crosslinking and remove protein, proteinase K was then added to a final concentration of 0.8 mg/mL and the samples were incubated for 30 minutes at 42°C and then an additional 6 hours at 65°C. 300 µL QG buffer and 100 µL of isopropanol was then added to each sample and a magnetic stand was used to pellet the Dynabeads. The supernatant was then collected and DNA was isolated using QIAquick gel extraction kit (Qiagen) according to the manufacturer’s instructions. DNA concentration was determined using the Nanodrop 8000 (Thermofisher Scientific) and sample were stored at -80°C.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NovaSeq 6000
 
Data processing ChIP-seq reads were aligned to the mm10 genome using Bowtie2 version 2.4.2.
Peaks were called using MACS3 version 3.0.0a7 with parameters -f BAM -g mm -B -q 0.01 –broad. Alignment files InputAB* were used as control conditions for H3K27me3 and H3K4me2 ChIP-Seq peaks. Alignment files InputD* were used as control conditions for KDM6A ChIP-Seq peaks. Alignment files InputB* were used as control conditions for H3K4me1 ChIP-Seq peaks.
Assembly: mm10
Supplementary files format and content: bam
Supplementary files format and content: bigwig
Supplementary files format and content: broadPeak
 
Submission date Apr 03, 2023
Last update date Jun 08, 2023
Contact name Matthew Oser
Organization name Dana Farber Cancer Institute
Department Thoracic Oncology
Street address 360 Longwood Avenue
City Boston
State/province MA
ZIP/Postal code 02215
Country USA
 
Platform ID GPL24247
Series (2)
GSE211165 KDM6A Epigenetically Regulates Subtype Plasticity in Small Cell Lung Cancer
GSE228347 KDM6A Epigenetically Regulates Subtype Plasticity in Small Cell Lung Cancer
Relations
BioSample SAMN34062202
SRA SRX19859551

Supplementary file Size Download File type/resource
GSM7141043_InputB_sgKDM6A_236R.bw 424.5 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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