|
| Status |
Public on Apr 01, 2022 |
| Title |
DamOnly - 15-somite embryo (KIN5600_index04) |
| Sample type |
SRA |
| |
|
| Source name |
15-somite zebrafish embryo
|
| Organism |
Danio rerio |
| Characteristics |
developmental stage: 15-somite embryo
dam-fusion protein: untethered Dam
molecule subtype: genomic DNA; mRNA
|
| Treatment protocol |
Tübingen longfin (WT) pairs were set up and the following morning, approximately 1nL of 1ng/ul Dam-Mphosph8 mRNA or 0.5ng/ul Dam-GFP mRNA was injected into the yolk at the 1 cell stage. Embryos were slowed down overnight at 23oC and the following morning all embryos were manually dechorionated.
|
| Growth protocol |
Adult zebrafish (Danio Rerio) were maintained and embryos raised and staged as previously described (PMID: 31510859).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
At 15-somite stage, embryos were transferred to 2ml Eppendorf tubes and digested with 0.1% Collagenase type II from Cl. Histolyticum (Gibco) in Hanks Balanced Salt Solution without Mg2+/Ca2+ (Thermofisher) for 20-30 mins at 32oC with constant shaking. Once embryos were noticeably digested, cell solution was spun at 2000g 5mins at room temperature and the supernatant was removed. Cell pellet was resuspended with TrypLE Express (Thermofisher) and digested for 10mins at 32oC with constant shaking. Cell solution was inactivated with 10% Fetal Bovine Serum (Thermofisher) in Hanks Balanced Salt Solution without Mg2+/Ca2+ and filtered through a 70um cell strainer (Greiner BIo-One). Cells were pelleted at 2000g 5min room temperature and washed twice with 10% Fetal Bovine Serum (Thermofisher) in Hanks Balanced Salt Solution without Mg2+/Ca2+. Hoescht at a final concentration of 16.8ug/ml was added to the cell solution and incubated for 30mins at 28oC in the dark. Solution was then filtered through a 40um cell strainer (Greiner BIo-One), Propidium iodide was added at a final concentration of 5ul/ml and immediately sorted on a FACS Influx (BD) gating for alive cells in the G2M phase into 384well plates containing primer solution.
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 |
GPL30614 |
| Series (2) |
| GSE184035 |
Single-cell profiling of histone modifications and transcription in developmental systems with EpiDamID [zebrafish] |
| GSE184036 |
Single-cell profiling of histone modifications and transcription in developmental systems with EpiDamID |
|
| Relations |
| BioSample |
SAMN21417426 |
| SRA |
SRX12163750 |
