|
| Status |
Public on Jul 01, 2019 |
| Title |
BIR_pol1YA_rnh202_24hr_NT_2 |
| Sample type |
SRA |
| |
|
| Source name |
BIR_pol1YA_rnh202_24hr_NT
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype/variation: pol1-Y869A rnh202{delta}
growth phase: Log phase cell culture
|
| Growth protocol |
Yeast strains were grown to log phase at 30°C in YPR medium before treated with 0.1 μg/ml doxycycline, 20 μg/ml nocodazole for 2 hours, followed by 50 μg/ml doxycycline and 2.5 mM IAA for 1 hours before HO induction. Samples were taken at various time points.
|
| 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 PmeI 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 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.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
TAK_2054
TAK_2054-BIR_pol1YA_rnh202_24hr_NT
processed data file: TAK_2038_2054-BIR_pol1YA_rnh202_24hr_NT_after_reverse_normalized.bw
processed data file: processed data file: TAK_2038_2054-BIR_pol1YA_rnh202_24hr_NT_after_reverse_normalized.bw
|
| 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 sequence (Ch_10_before_BIR.fa/Ch_10_after.fa), 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 (bowtie 1.2 -m1 -v2)
Retain 5' end only for all single- and paired-end unique alignments, combine
Determine counts of ends mapped to PmeI restriction sites, calculate size factors using method applied in DESeq, Anders et al., 2010
Filter ends mapping within 2 nt of an PmeI restrition site or site with 1 bp mismatch relative to PmeI motif, divide by previously determined size factors
Genome_build: Ch_10_before_BIR.fa, Ch_10_after_BIR.fa
Supplementary_files_format_and_content: bigWig, count of HydEn-seq 5' ends per position
|
| |
|
| Submission date |
Jun 30, 2019 |
| Last update date |
Jul 07, 2019 |
| Contact name |
Zhi-Xiong Zhou |
| E-mail(s) |
zhouzhixiong2013@gmail.com
|
| Organization name |
National Institute of Envinromental Health Sciences
|
| Street address |
111 TW Alexander Dr.
|
| City |
Durham |
| ZIP/Postal code |
27709 |
| Country |
USA |
| |
|
| Platform ID |
GPL21656 |
| Series (1) |
| GSE133558 |
DNA polymerase Δ synthesizes both strands during break-induced replication |
|
| Relations |
| BioSample |
SAMN12168011 |
| SRA |
SRX6384820 |
