|
| Status |
Public on Jun 14, 2012 |
| Title |
Kc WT Late S-Phase Repliseq Rep2 extraction6_seq1 aliquote 1 |
| Sample type |
SRA |
| |
|
| Source name |
Kc WT Late S-Phase Repliseq Rep2
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: embryo-derived cell-line
developmental stage: late embryonic stage
genotype: se/e
Sex: Female
|
| Growth protocol |
The general growth protocol for growing Drosophila cell lines in culture.
Cells are pulse labeled with 50ug/ml BrdU for one hour, and washed with 1x PBS.
FACS Protocol.1. Spin cells down and resuspend in 300µl of PBS. (For large pellets increase this volume and increase the EtOH volume proportionally)2. Fix by adding 5ml of 70% EtOH Dropwise while vortexing gently, incubate at -20ºc for at least 30 minutes.3. Spin, remove EtOH and resuspend in 5ml PBS. Allow cells to rehydrate at 4ºc for at least an hour.4. Spin and resuspend the cells in 4ml of PBS with 10µg/ml RNaseA and 10µg/ml PI. Incubate at RT for at least 30min up to several hours (alternatively incubate for 10min at 37ºc)Cells were sorted into early mid and late S-phase fractions using a FACS Vantage SE (BD Biosciences) equipped with the DiVa acquisition system.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
An immunoprecipitation procedure in which anti-BrdU is used to enrich for BrdU labeled DNA. We have a population of cells that have been labeled with BrdU and sorted into different stages of S-phase using FACS for replication timing analysis. To isolate the BrdU-substiluted DNA from these cells, we must immunoprecipitate them with an anti-BrdU antibody. This method was optimized and selected from two papers - Cimbora (MeS,2000) and Hansen (Cell, 1993). Generation of genomic library suitable for Illumina sequencing starts with random shearing of genomic DNA with sonication followed by Illumina-developed sample preparation protocol.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Data processing |
Illumina_sequencing:DM:1 protocol. Load sample onto flow cell at a (usually 2-8 pM, variable) concentration and run on an Illumina Genome Analyzer II for single-end sequencing for 36 or 76 nt. Image data is then deconvoluted using the most current versions of Firecrest, Bustard, and Gerald (or equivalents). Processed data are obtained using following parameters: read length is 36 Illumina_seq_maq_processing:DM:1 protocol. Using the MAQ command-line tool v.0.7.1, reads are converted from Illumina FASTQ to Sanger FASTQ to Binary FASTQ. The Drosophila genome v.5.1 is then converted into binary fasta format. Then the reads are mapped back to the Drosophila genome using maq 0.7.1 with a maximum of 3 mismatches per read and a mapview file is created which is then read into a local relational database. Processed data are obtained using following parameters: genome version is 5.1 Illumina sequencing WIG Generation RPKM:DM:1 protocol. For Repliseq experiments, a bin size of 10000 bp stepping every 2500 bp was used.
|
| |
|
| Submission date |
Jun 14, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
DCC modENCODE |
| E-mail(s) |
help@modencode.org
|
| Phone |
416-673-8579
|
| Organization name |
Ontario Institute for Cancer Research
|
| Lab |
modENCODE DCC
|
| Street address |
MaRS Centre, South Tower, 101 College Street, Suite 800
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 0A3 |
| Country |
Canada |
| |
|
| Platform ID |
GPL9058 |
| Series (1) |
|
| Relations |
| SRA |
SRX154746 |
| BioSample |
SAMN01054420 |
| Named Annotation |
GSM948524_DM71.bed.gz |
