|
| Status |
Public on Apr 03, 2023 |
| Title |
Lgr5-tdT rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Lgr5-tdT
|
| Organism |
Mus musculus |
| Characteristics |
cell line: Lgr5-tdT
cell type: flow sorted intestinal epithelial cells
|
| Growth 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 1:3?1:5 split ratio.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Flow-sorted tdT+ cells (25,000 cells in duplicates) were lysed to prepare nuclear pellets which then underwent transposition with TDE1 Enzyme (Illumina, 20034197). Tagmented DNA was purified using Zymo DNA Clean and Concentrator-5 Kit (cat# D4014) and the purified DNA was PCR amplified with NEBNext 2x MasterMix and Illumina adapters (table S4). The libraries were purified post-PCR using AMPure XP beads (Beckman Coulter). 150-bp paired-end reads were sequenced on a Novaseq instrument (Illumina).
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
TD1E2_CKDL220002467-1a_H7VGVDSX3_L2
|
| Data processing |
ATAC data processing
ATAC sequencing data was analyzed as described previously (PMID: 28648363). In brief, reads were mapped to the mm10/GRCm38 genome using Bowtie2 aligner version 2.3.5.ßMACS2 was used to call peaks using the parameters µ-t $bamfile -f BAMPE -n qc/macs/$base.macs2 -q 0.01 -g mm?.ßBigwig files were generated using deepTools bamCoverage with the options µbamCoverage --binSize 10 --smoothLength 30 -p 4 --normalizeUsing RPGC --effectiveGenomeSize 2730871774 --extendReads $fragLength -b $file -of bigwig? (PMID: 24799436).ßßAll bigwig and bed files were filtered using the ENCODE Blacklist. Only peaks with P-value < 0.00001 were considered for further analyses. Proximal peaks (-2kb to 2kb relative to TSS) called by macs2 were linked/annotated to genes by ChIPseeker version 1.34.1. Then, computeMatrix and plotHeatmap functions from deepTools were used to visualize peaks.ß
Assembly: mm10/GRCm38
Supplementary files format and content: bigwig
|
| |
|
| Submission date |
Dec 19, 2022 |
| Last update date |
Apr 03, 2023 |
| Contact name |
Nilay Sethi |
| E-mail(s) |
nilay_sethi@dfci.harvard.edu
|
| Organization name |
DANA-FARBER CANCER INSTITUTE
|
| Street address |
450 Brookline Ave
|
| City |
BOSTON |
| State/province |
Massachusetts |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE221298 |
Aberrant cell state plasticity mediated by developmental reprogramming precedes colorectal cancer initiation [ATAC-seq] |
| GSE221300 |
Aberrant cell state plasticity mediated by developmental reprogramming precedes colorectal cancer initiation |
|
| Relations |
| BioSample |
SAMN32308851 |
| SRA |
SRX18769917 |
