|
| Status |
Public on Apr 19, 2024 |
| Title |
RPE-1 hTERT FUCCI bulk single pulse 120min early S |
| Sample type |
SRA |
| |
|
| Source name |
Retinal Pigment Epithelial
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Retinal Pigment Epithelial
cell line: RPE-1 hTERT FUCCI
treatment: DMSO
genotype: WT
|
| Treatment protocol |
Cells were treated with indiicated inhibitors for diverse periods of time
|
| Growth protocol |
RPE-1 hTERT FUCCI or XRCC1 KO were maintained in DMEM/F12, 10% FCS and 1% Pen-Strep
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
TrypLE digestion
Custom library protocol. Briefly, EdU was clicked to Biotin-PEG3-Azide. Single cells were sorted in a 384-well plate. Subsequently, cells were lysed wioth ProtK and the genome was digested with NLAIII. Next, DNA fgragments were end-repaired by Klenow Large Fragment and PNK. On these repaired ends we ligated a custom adapter sequence followed by affininty purification of EdU-Biotin miolecules with magnetic streptadin beads. Non-modified strands were recovered by heat denaturation and the complimetary strand was re-generated by a primer extension reaction. Finally, libraries were amplified by a combination of T7 in vitro trasncription, reverse transcription and polymerase chain reaction containing Illumina handles
Illumina RPI small RNA adapters
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
bulk EdUseq library
|
| Data processing |
demultiplexing reads; extracting cell barcodes from reads
trimming adapter sequences with cutadapt
mapping to human genome with bwa -mem algorithm
custom bam tagging tool to add flags which we later select for
extracting reads wtih specific bam flags and write to tsv.gz file
Assembly: GRCh38.p12 / Ensembl release 97 for Homo Sapiens
Supplementary files format and content: Data table containing cell name, chromosome, read start, length, allele
Library strategy: DNA-seq
|
| |
|
| Submission date |
Aug 11, 2022 |
| Last update date |
Apr 19, 2024 |
| Contact name |
Jeroen van den Berg |
| E-mail(s) |
[email protected]
|
| Phone |
+31610597913
|
| Organization name |
Hubrecht Institute
|
| Lab |
van Oudenaarden lab
|
| Street address |
Uppsalalaan 8
|
| City |
Utrecht |
| State/province |
Utrecht |
| ZIP/Postal code |
3584 CT |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE211037 |
Quantifying DNA replication speeds in single cells by scEdU-seq |
|
| Relations |
| BioSample |
SAMN30265191 |
| SRA |
SRX17033184 |
