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| Status |
Public on Jan 30, 2019 |
| Title |
TAK_1945-S_pombe_HydEn-seq_cdc20-M630F_rnh201_RNaseHII |
| Sample type |
SRA |
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| Source name |
Log phase cell culture
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: cdc20-M630F rnh201delta
treatment: RNaseHII
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| Growth protocol |
Yeast strains were grown to mid log phase (OD600=0.5-1) at 30°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.
HydEn-seq libraries are constructed as previously described (Clausen, et al. 2015 NSMB) with modifications by the following steps. Briefly, extract yeast genomic DNA (gDNA) using Epicenter MasterPure Yeast DNA Purification Kit. Treat 200 ng gDNA with Shrimp Alkaline Phosphatase (rSAP, New England Biolabs). Denature rSAP and restriction digest with SbfI-HF at 37 °C for 1 hour. Split the DNA into two fractions. Treat one fraction with RNase HII (NEB) at 37 °C for 2 hours and designate the other fraction as non-treatment control. Denature the DNA by incubation at 90 °C for 2 min and ligate to adaptor ARC140 by T4 RNA ligase 1 (NEB) overnight at 25 °C. Denature DNA and anneal to the duplex adaptor ARC76/77. Synthesize the second strand using T7 DNA polymerase (NEB). Amplify the library for 20 cycles by primer ARC49 and an indexing primer using KAPA HiFi HotStart Ready Mix (KAPA Biosystems). 0.8 volumes of MagBio HighPrep PCR beads were used to purify DNA between steps that require changing buffers and/or removal of oligo adaptors and in the final library cleanup. The library was sequenced on an Illumina Illumina HiSeq 4000 machine for paired end 50 bp reads. The rSAP, SfbI-HF and RNase HII treatments are the main modifications to the original protocol to improve on signal-to-noise ratio, quantitation and specificity.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Library strategy: HydEn-seq
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
Align end 1 to index of oligos used during library prep, exclude successful pairs from further analysis (bowtie 1.2 -k1 -v2, see supplementary file oligos.fa)
Align remaining pairs to 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 reference genome (bowtie 1.2 -m1 -v2)
Retain 5' end only for all single- and paired-end unique alignments, combine
Determine counts of ends mapped to SbfI-HF restriction sites, calculate size factors using method applied in DESeq, Anders et al., 2010
Filter ends mapping within 2 nt of an SbfI-HF restrition site or site with 1 bp mismatch relative to SbfI-HF motif, divide by previously determined size factors
Genome_build: L03 background S. cerevisiae assembly (see supplementary files L03_ref_v2.fa), S. pombe assembly 2.23
Supplementary_files_format_and_content: bigWig, count of HydEn-seq 5' ends per position
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| Submission date |
Jan 29, 2019 |
| Last update date |
Feb 04, 2019 |
| Contact name |
Zhi-Xiong Zhou |
| E-mail(s) |
[email protected]
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| Organization name |
National Institute of Envinromental Health Sciences
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| Street address |
111 TW Alexander Dr.
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| City |
Durham |
| ZIP/Postal code |
27709 |
| Country |
USA |
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| Platform ID |
GPL22682 |
| Series (1) |
| GSE125855 |
Mapping polymerase usage by tracking ribonucleotide incorporation (HydEn-seq) during DNA replication |
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| Relations |
| BioSample |
SAMN10841434 |
| SRA |
SRX5307203 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3583289_TAK_1945-S_pombe_HydEn-seq_cdc20-M630F_rnh201_RNaseHII_norm_forward.bw |
6.7 Mb |
(ftp)(http) |
BW |
| GSM3583289_TAK_1945-S_pombe_HydEn-seq_cdc20-M630F_rnh201_RNaseHII_norm_reverse.bw |
6.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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