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| Status |
Public on Apr 03, 2023 |
| Title |
Adenoma, IgG |
| Sample type |
SRA |
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| Source name |
Adenoma organoids derived from a patient with Familial Adenomatous Polyposis syndrome
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| Organism |
Homo sapiens |
| Characteristics |
cell line: Adenoma organoids derived from a patient with Familial Adenomatous Polyposis syndrome
cell type: Colon epithelium, Adenoma
chip antibody: IgG, Epicypher
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| Treatment protocol |
Colonic glands were isolated, treated with EDTA, and then resuspended in 30-50 ul of Matrigel (BD Bioscience) and plated in 24-well plates. WNT/R-spondin/Noggin (WRN) containing DMEM/F12 with HEPES (Sigma-Aldrich) containing 20% FBS, 1% penicillin/streptomycin and 50ng/ml recombinant mouse EGF (Life Technologies) was used for culturing ApcKO colon organoids. For the first 2-3 days after seeding, the media was also supplemented with 10 mM ROCK inhibitor Y-27632 (Sigma Aldrich) and 10 mM SB431542 (Sigma Aldrich), an inhibitor for the transforming growth factor (TGF)-? type I receptor to avoid anoikis. For passage, colon organoids were dispersed by trypsin-EDTA and transferred to fresh Matrigel. Passage was performed every 3-4 days with a 1:3û1:5 split ratio. For human colon organoid culture, the previous media was supplemented with antibiotics 100 ug/ml Primocin (Invivogen), 100 ug/ml Normocin (Invivogen); serum-free supplements 1X B27 (Thermo Fisher (Gibco)), 1X N2 (Thermo Fisher (Gibco)); chemical supplements 10mM Nicotinamide (Sigma), 500mM N-acetylcysteine (Sigma), hormone 50mM [Leu15]-Gastrin (Sigma), growth factor 100ug/ml FGF10 (recombinant human) ( Thermo Fisher) and 500nM A-83-01 (Sigma), which is an inhibitor of the TGF-B Receptors ALK4, 5, and 7.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
CUT&RUN for SOX9 and IgG control in Adenoma and Normal organoids was done using CUTANA ChIC/CUT&RUN kit (Epicypher #14-1048) following manufacturerÆs protocol. Briefly, 5???105 cells were captured with Concanavalin A, permeabilized using 0.01% digitonin and incubated with 0.5?g antibody (anti-Sox9 (CST, #82630) and IgG (# 13-0042; Epicypher) in 50?L antibody buffer (20mM HEPES at pH 7.5, 150mM NaCl, 0.5mM Spermidine, 1x protease inhibitor cocktails (EDTA-free tablet; Roche), 2 mM EDTA, 0.01% digitonin) overnight. After removing unbound antibody, pAG-MNase (20X) in 50?L cell permeabilization buffer was added to the cells and incubated for 10 min at RT. Then CaCl2 (2mM) was added to activate MNase and incubated at 4??C for 2 hr. The reaction was stopped using 33?L of 2X STOP buffer and E. coli spike-in DNA (0.5ng) was added as a control. The DNA from the released chromatin in the supernatant was purified and then quantified using Qubit dsDNA HS assay kit (Agilent Technologies). CUT&RUN libraries were constructed using NEBNext Ultra II DNA library preparation kit as described previously (Skene et al., 2018) with a few modifications. Briefly, end repair and dA-tailing were conducted on 6?ng of CUT&RUN eluted DNA for 30 min at 20?C followed by 1hr at 50?C. After adaptor ligation for 30 min at 20?C, the DNA fragments were purified by 1.75X vol of AMPure XP beads (Beckman Coulter) followed by 10-12 cycles of PCR amplification with Next Ultra II Q5 master mix. The PCR products were purified with 1X vol of AMPure XP beads. After quantitative and qualitative analysis, libraries with different indexes were pooled and sequenced on Illumina Novaseq platform with paired-end 150-bp reads.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
AD3_IgG
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| Data processing |
CUT&RUN sequencing data was analyzed as described previously(71), following the standard pipeline (https://yezhengstat.github.io/CUTTag_tutorial/index.html). In brief, paired-end 150-bp reads were aligned to GRCh38 human genome using Bowtie2 version 2.2.5(72) with options: --local --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700. Technical replicates (n = 4 per condition) were merged before reads alignment to increase the power of peak calling. macs2(73) was used to call peaks from bam files. For SOX9 CUT&RUN peak calling, parameters ùt input_file ûp 1e-5 ûf BAM ûkeep-dup all ûn out_name was used to call narrow peaks. To check the SOX9 binding profile and enhancer activity of specific gene sets, proximal peaks (-2kb to 2kb relative to TSS) called by macs2 were linked/annotated to genes by ChIPseeker version 1.34.1(74). Then, computeMatrix and plotHeatmap functions from deepTools(75) were used to visualize peaks.
Assembly: hg38
Supplementary files format and content: bigwig
Library strategy: CUT&RUN
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| Submission date |
Dec 19, 2022 |
| Last update date |
Apr 03, 2023 |
| Contact name |
Nilay Sethi |
| E-mail(s) |
nilay_sethi@dfci.harvard.edu
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| Organization name |
DANA-FARBER CANCER INSTITUTE
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| Street address |
450 Brookline Ave
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| City |
BOSTON |
| State/province |
Massachusetts |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE221299 |
Aberrant cell state plasticity mediated by developmental reprogramming precedes cancer initiation [CUT&RUN] |
| GSE221300 |
Aberrant cell state plasticity mediated by developmental reprogramming precedes colorectal cancer initiation |
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| Relations |
| BioSample |
SAMN32309237 |
| SRA |
SRX18769899 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6857978_AD3_IgG.bigwig |
330.2 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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