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| Status |
Public on Feb 27, 2018 |
| Title |
TAK1082_pol3L612M_pol2-16 |
| Sample type |
SRA |
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| Source name |
TAK1082 pol3L612M pol2-16
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: pol3L612M pol2-16
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| Growth protocol |
Yeast strains were grown to mid log phase (OD600=0.5-1) at 23°C in YPDA medium supplemented with 0.25 mg/ml adenine
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was isolated using the MasterPure™ Yeast DNA Purification Kit (Epicentre) without RNase A treatment.
Ribonucleotides were mapped in the genomic DNA using Hydrolytic End Sequencing technique (HydEn-seq) described by Clausen et al., 2015. Briefly, DNA was isolated from exponentially growing yeast (OD600: 0.5 – 1/ ml) with using a kit for genomic DNA isolation from yeast (Epicenter). Libraries for Next Generation Sequencing were prepared with using 1 µg of DNA, which was treated with 20 U of restriction enzyme SbfI-HF (New England Biolabs) at 37o C for 1h. Next DNA was treated under 300 mM KOH at 55o C for 2 hours and precipitated with ethanol. Library were constructed as described in Clausen et al., 2015. Briefly, DNA was denatured for 3 min at 85o C, phosphorylated with 10 U of T4 PNK (#M0236, New England Biolabs) and purified with 1.8 volume of magnetic beads (Magbio). After denaturation for 3 min at 85o C, DNA fragments were ligated with adapter ARC140 over night at 25o C using 10 U of T4 RNA ligase (#M0204, New England Biolabs). This was followed by DNA purification with magnetic beads and the second strand synthesize using 4 U of T7 DNA polymerase (#M0274, New England Biolabs) and ARC76/77 duplex. After DNA purification with magnetic beads, unique indexes were added to DNA fragments using KAPA HiFi HotStart Ready Mix (#KK2602, KAPA Biosystems). The library concentrations and sizes were determined using Bioanalyzer. Libraries were pooled and subject for paired end sequencing with Illumina HiSeq 2500.
HydEn-seq
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Base calling and generation of FASTQ files performed by Illumina Hiseq standard pipeline
Reads trimmed for adapter sequence using cutadapt 1.12, discarding pairs with one or both reads shorter than 15 nt
Combine technical replicates
Align end 1 to index of oligos used during library prep, exclude successful pairs from further analysis (bowtie 1.2 -k1 -v2)
Align remaining pairs to W303 reference genome, trim single nt from 3' end of reads to allow mapping of 100% overlapping pairs (bowtie 1.2 -m1 -v2 -X10000 -3 1)
Align end 1 of unmapped pairs to W303 reference genome (bowtie 1.2 -m1 -v2)
Retain 5' end only for all single- and paired-end unique alignments, shift 1 base upstream and combine
Genome_build: W303 background RNR1 assembly (see supplementary files)
Supplementary_files_format_and_content: bigWig, count of HydEn-seq 5' ends per position
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| Submission date |
Jul 20, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Marta Anna Garbacz |
| E-mail(s) |
garbaczmaw@gmail.com
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| Phone |
9195410268
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| Organization name |
NIEHS/NIH
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| Department |
GISBL
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| Lab |
DNA Replication Fidelity Lab
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| Street address |
111 T.W. alexander Dr.
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| City |
Research Triangle Park |
| State/province |
NC |
| ZIP/Postal code |
27713 |
| Country |
USA |
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| Platform ID |
GPL17342 |
| Series (1) |
| GSE101698 |
Evidence that DNA polymerase δ contributes to initiation of leading strand DNA replication in Saccharomyces cerevisiae |
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| Relations |
| BioSample |
SAMN07373284 |
| SRA |
SRX3022285 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2712355_TAK1082_pol3L612M_pol2-16_forward.bw |
4.9 Mb |
(ftp)(http) |
BW |
| GSM2712355_TAK1082_pol3L612M_pol2-16_reverse.bw |
4.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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