|
| Status |
Public on Sep 01, 2017 |
| Title |
Chlamydomonas reinhardtii, replicate 2 (4A+, 48h post Nitrogen depletion) |
| Sample type |
SRA |
| |
|
| Source name |
Chlamydomonas reinhardtii, replicate (4A+, 48h post Nitrogen depletion) _Whole organism
|
| Organism |
Chlamydomonas reinhardtii |
| Characteristics |
strain: 4A+
treatment: 48h post Nitrogen depletion
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
C. reinhardtii wild type strain 4a+ were cultured using Tris-acetate-phosphate (TAP) medium in Erlenmeyer flasks at 25 degrees Celsius with agitation at 180 rpm under light irradiation at 100 microE. Cells were washed twice with nitrogen-free media and re-suspended to a density of 2 x 106 cells/ml and harvested at 48 hr post starvation. Cells were lysed using lysis buffer (50mM Tris-HCl pH 8, 200mM NaCl, 2mM EDTA, 2% SDS and proteinase K) and genomic DNA was extracted using CTAB-based protocol39.
5-10ug of gDNA was sheared to >10kb using g-tube (Covaris). The sheared DNA was treated with DNA damage repair mix followed by end repair and ligation of SMRT hairpin adapters using SMRTbell Template Preparation Reagent Kit (Pacific Biosciences). Fragments without adaptors are digested with exonuclease. Libraries were sequenced on Pacific Biosciences RSII sequencer using standard protocols.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
PacBio RS II |
| |
|
| Data processing |
SMRT sequencing reads were processed and mapped to the respective reference genome sequences using the SMRTPortal Analysis platform, v.2.3.0 with BLASR mapping algorithm using the standard mapping protocol and the modifications protocol using default parameters.
Genome_build: C.Reinhardtii_236 (JGI Phytozome assembly V5.0)
The pipeline used to detect the modifications is called “DNA Modification Detection with SMRT Sequencing using R” and can be found at https://github.com/PacificBiosciences/kineticsTools. Interpulse durations (IPDs) were measured for all pulses aligned to each position in the reference sequence. The format of the output CSV/GFF files with modifications is described at http://www.pacb.com/pdf/TN_Detecting_DNA_Base_Modifications.pdf. The CSV file contains statistical analysis of each position in the reference. The GFF file includes sequence contexts only for sites of putative modification defined as positions with p-values of 0.01 or less.
Supplementary_files_format_and_content: GFF and CSV files [Chlamy.modifications.48H.Rep1and2.gff.gz, Chlamy.modifications.48H.Rep1and2.csv.gz]
|
| |
|
| Submission date |
May 13, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Denis Tolkunov |
| Organization name |
DOE Joint Genome Institute
|
| Street address |
2800 Mitchell Dr
|
| City |
Walnut Creek |
| State/province |
CA |
| ZIP/Postal code |
94598 |
| Country |
USA |
| |
|
| Platform ID |
GPL20195 |
| Series (1) |
| GSE68860 |
Genome-wide single-molecule sequencing of 6-methyladenine in eukaryotes |
|
| Relations |
| BioSample |
SAMN03657258 |
| SRA |
SRX1026805 |
