|
| Status |
Public on Apr 01, 2022 |
| Title |
DamOnly - EB and ESC day14 (KIN5599_index06) |
| Sample type |
SRA |
| |
|
| Source name |
Embryoid Bodies and 2i-cultured ESC (day 14)
|
| Organism |
Mus musculus |
| Characteristics |
genotype: 129S1/SvImJ x CAST/Eij
dam-fusion protein: untethered Dam
molecule subtypes: genomic DNA; mRNA
|
| Treatment protocol |
Two days before single cell sorting the EB’s are shifted to CM+/- medium with 1mM IAA, and before single cell sorting on day 7, 10 and 14 the Dam-POI constructs are induced in the following way: 6hrs no IAA (Ring1B), 20hrs no IAA + 7hrs 1uM 4-OH Tamoxifen (Dam-ScFv-H3K27me3) and 7hrs no IAA + 4hrs 1uM 4-OH Tamoxifen (Dam-only), and the best developed embryonic bodies were handpicked and pooled together.
|
| Growth protocol |
For the EB differentiation the stable knockin F1ES lines were cultured for 2 weeks on 0.1% gelatin coated plates in 2i ES cell culture medium: 48% DMEM/F12 (Gibco) and 48% Neurobasal (Gibco), supplemented with 1x N2 (Gibco), 1x B27 supplement + vitA (Gibco), 1x non-essential amino acids, 1% FBS, 1% Pen/Strep, 0.1mM β-mercaptoethanol, 1 μM PD0325901 (Axon Medchem, PZ0162-5MG), 3 μM CHIR99021 (Tocris, SML1046-5MG) and 10^3U/ml ESGRO mLIF medium before plating for EB differentiation according to ATCC protocol. On day1 of the differentiation 2x10^6 cells are grown in suspension on a non-coated bacterial 10cm dish with 15ml CM +/- (with β-mercaptoethanol without LIF) and 0.5mM IAA. The next day half the cell suspension is divided over 5 non-coated bacterial 10cm dishes with 15ml CM+/- medium and 0.5mM IAA and plates are refreshed every other day.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were trypsinized and harvested in medium with 20 μg/mL Hoechst 34580 (Sigma 63493) per 1x10^6 cells for 45 minutes at 37°C. The cells were stained for live/dead gating with 1ug/ml propidium iodide and singlets were index sorted based on their Hoechst profile. One cell per well was sorted into 384-well plates (Biorad, HSP3801) using the BD FACS Jazz cell sorter. Wells contained 5 μL filtered mineral oil (Sigma) and 50nL of 0.5uM unique CELseq primer.
The scDam&T protocol and library preparation was performed as previously described in detail (PMID: 32350457).
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| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
| |
|
| Description |
Raw data files include both DamID and CEL-seq reads
|
| Data processing |
Library strategy: scDam&T-seq
Libraries are demultiplexed based on the sample-specific and readout-specific barcode present in R1, using custom pipelines (available at: https://github.com/KindLab/scDamAndTools).
DamID alignment: DamID reads are aligned using bowtie2 (v. 2.3.3.1) with the following parameters: “--seed 42 --very-sensitive -N 1”.
DamID filtering: Alignments with a MAPQ < 10 are removed.
DamID procesing: Using our own custom pipeline (see KindLab GitHub), aligned reads are matched to known GATC positions in the genome to get the number of observations per (strand-specific) GATC position.
DamID processing: GATC-position counts are deduplicated using UMI information to allow a maximum of 4 unique UMIs per GATC position.
DamID processing: Unique GATC counts are summarized in genomic intervals at the desired resolution.
CELseq alignment: CELseq reads are aligned using GRCm38 (v. 89) transcript models and tophat2 (v. 2.1.1) with the following paramters: : “--segment-length 22 --read-mismatches 4 --read-edit-dist 4 --min-anchor 6 --min-intron-length 25 --max-intron-length 25000 --no-novel-juncs --no-novel-indels --no-coverage-search --b2-very-sensitive --b2-N 1 --b2-gbar 200”.
CELseq filtering: Alignments with a MAPQ < 10 are removed.
CELseq processing: Using our own custom pipeline (see KindLab GitHub), we assign alignments to genes using a method similar to htseq-count with mode "intersection_strict".
CELseq processing: Transcript counts are deduplicated using UMI information, allowing an unlimited number of UMI-unique counts per gene.
Genome_build: mm10
Supplementary_files_format_and_content: celseq count table: count table containing UMI-unique transcript counts per gene for all single-cell samples; damid count table: count table containing UMI-unique GATC counts per 10-kb interval for all single-cell samples; sample annotation: table containing detailed sample information for all single-cell samples; sample barcodes: sample-specific and readout-specific barcodes that can be used to demultiplex the raw data. Barcodes follow a 3-NNN-3-NNN format, where "N" positions indicate the barcode-specific sequence, and the "3" indicates positions of a 3nt UMI sequence.
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| |
|
| Submission date |
Sep 13, 2021 |
| Last update date |
Apr 01, 2022 |
| Contact name |
Jop Kind |
| E-mail(s) |
j.kind@hubrecht.eu
|
| Organization name |
Hubrecht Institute
|
| Street address |
Uppsalalaan 8
|
| City |
Utrecht |
| ZIP/Postal code |
3584CT |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL30172 |
| Series (2) |
| GSE184033 |
Single-cell profiling of histone modifications and transcription in developmental systems with EpiDamID [mouse EB] |
| GSE184036 |
Single-cell profiling of histone modifications and transcription in developmental systems with EpiDamID |
|
| Relations |
| BioSample |
SAMN21417076 |
| SRA |
SRX12162951 |
