|
| Status |
Public on Oct 28, 2020 |
| Title |
FACS-sorted intestinal embryonic blastomeres DamID GFP control, replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
Intestinal embryonic blastomeres
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
developmental stage: Embryonic blastomeres
tissue: Intestine
sample type: FACS-sorted
strain: IS3653
expressing: Dam fusion to GFP
|
| Growth protocol |
Synchronized gravid adult hermaphrodites containing 8-10 eggs were treated with hypochlorite. Eggs were then incubated for 3 hours in M9 at 25°C until they reached the 1.5 fold stage. Eggs were then transferred to 500 μl egg buffer and pelleted 1 min at 2000 rpm. The supernatant was then aspirated, leaving 100 μl of buffer with the pellet. 500 U of chitinase (Sigma-Aldrich;C8241-25U) were added and the mixture resuspended and further incubated for one hour at room temperature. Chitinase was then neutralized with 800 μl Leibovitz medium. Digested embryos were then recovered by centrifugation at 3000 rpm for 5 minutes at 4°C. Embryos were then dissociated into isolated blastomeres by pipetting up and down with a P1000 pipette, up to 150 times until dissociation was complete. The cell population was then filtered with a Millex-SV syringe 5 μm filter (Millipore;SLSV025LS). 500 fluorescent cells were sorted using a BD FACSAria Fusion (8000 events/second; 85µm nozzle) in sterile PCR tubes containing 1ul of pick buffer (50mM Tris-HCl pH 8.3; 75mM KCl; 3mM MgCl2; 137mM NaCl). Collected samples were then frozen in liquid nitrogen before further processing. After DamID processing and PCR (see below), two technical replicates were pooled for sequencing library preparation.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For sorted cells, frozen samples were lysed by addition of 2 ul of lysis buffer (10mM TrisAc, 10 mM MgAc, 50 mM KA, 0.67% Tween 20, 0.67% Igepal + 1mg/ml Proteinase K) and incubated during 2 hours at 60ºC before Proteinase K inactivation at 95ºC for 15 minutes. DamID amplicons were obtained as previously described (Gomez-Saldivar, Meister, and Askjaer 2016) using 30 PCR cycles.
DamID PCR amplicons were therefore sequenced using nanopore sequencing. After AMPure XP (Beckman-Coulter) purification with 1.8 beads volume, DamID PCR amplicons were directly used for nanopore library barcoding and preparation using NBD-104 and LSK-109 kits (Oxford Nanopore).
Libraries were sequenced to obtain at least 1 million reads per library on MinION using flow cells R9.4.1.
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| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
MinION |
| |
|
| Description |
DamID amplicons from sorted intestinal embryonic blastomeres expressing Dam fusion to GFP
Intestine_Muscle_pass_barcode12.genes.details.csv
Intestine_Muscle_pass_barcode12.sorted_damid-vs-Dam.gatc.bedgraph
|
| Data processing |
Library strategy: DamID
Nanopore sequences were basecalled and demultiplexed using guppy 3.6 with the high accuracy model before mapping to the ce11 genome using minimap2 (Li 2018).
Reads were considered as DamID amplicons when both ends mapped +/- 8 bp from a genomic GATC motif. The bam files represent the filtered reads.
Filtered libraries were then used to call polymerase footprinting values using the damidseq_pipeline package (O. J. Marshall and Brand 2015) using bam files (parameters: --bamfiles --extend_reads=0 --rpm). The output of this step are bedgraphs.
Gene values were extracted using polii.gene.call (https://owenjm.github.io/damidseq_pipeline/) using WS270 genes definition. The output of this steps are csv files.
Genome_build: ce11
Supplementary_files_format_and_content: bam file with valid DamID reads
Supplementary_files_format_and_content: Normalized begraph (Dam fusion over Dam only)
Supplementary_files_format_and_content: Gene-level DamID values in csv format, with number of GATC per gene and FDR
|
| |
|
| Submission date |
Sep 03, 2020 |
| Last update date |
Oct 29, 2020 |
| Contact name |
Peter Meister |
| E-mail(s) |
peter.meister@unibe.ch
|
| Phone |
+41316844609
|
| Organization name |
University of Bern
|
| Department |
Institute of Cell Biology
|
| Lab |
Cell Fate and Nuclear Organization
|
| Street address |
Baltzerstrasse 4
|
| City |
Bern |
| State/province |
Schweiz |
| ZIP/Postal code |
3012 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL26522 |
| Series (2) |
| GSE157415 |
Tissue-specific transcription footprinting using RNA PolII DamID (RAPID) in C. elegans [Blastomeres] |
| GSE157418 |
Tissue-specific transcription footprinting using RNA PolII DamID (RAPID) in C. elegans |
|
| Relations |
| BioSample |
SAMN16049186 |
| SRA |
SRX9067970 |
