GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2258100

Query DataSets for GSM2258100

Status

Public on May 21, 2021

Title

RiSQ014_WT_CR_1_RH_rNMP

Sample type

SRA

Source name

MAT a G1 arrested yeast cells

Organism

Saccharomyces cerevisiae BY4741

Characteristics

taxonomy_id: 1247190
strain: YTT0003
mating type: MATa
genotype/variation: Parental WT yeast strain
cell cycle: G1-arrested by alpha factor
treatment group: RNaseH digestion

Growth protocol

Yeast strains were exponentially grown in two flasks of 500ml 1% Bacto Yeast Extract (BD), 2% Bacto Peptone and 2% Glucose (Wako) (YPD) or low glucose media with 0.05% Glucose (CR) around 1x10^7 cells/ml and arrested cell-cycle by addition of alpha-factor at final concentration 5 ug/ml for 2.5 hour at 30C with shaking (222 rpm) in INNOVA43 shaker.

Extracted molecule

genomic DNA

Extraction protocol

Yeast spheloplasts were lysed in pre-warmed lysis buffer (50 mM Tris-Cl pH7.5, 0.2 M NaCl, 0.1 M EDTA, 5% SDS) at 65°C by gentle pipetting and inversions. After phenol-chloroform extraction twice, nucleic acids were precipitated with twice volume of 99% ethanol, and the precipitates were rinsed with 70% ethanol. Dried nucleic acids were treated by RNase A (Wako) in 500 µl of RNase buffer (10 mM Tris-Cl pH7.5, 1 mM EDTA, 0.5 mg/ml RNase A) at 37°C for 2 h. After phenol-chloroform extraction twice, genomic DNAs were purified by ethanol precipitation.
RiSQ-seq was performed with sheared genomic DNAs. Ten µg of total DNA was sheared in microTUBE (Covaris) by Covaris M220 (peak power 140W, duty factor 10%, cycle/burst 200 and duration 1 minute). Agarose-gel purified DNA (400-450 bp range) was end-repaired by KAPA library preparation kit for Illumina (Kapa Biosystems) supplement with 2 mM ATP and 2.5 mM dNTP. One µg of the size-selected DNA and 25ng of spike-in standard cocktail were used for a primary library. Pre-primary library was prepared by KAPA library preparation kit and Illumina TruSeqHT-compatible adaptors (IDT). Free 3'-OH ends of the pre-primary library were masked by terminal deoxynucleotidyl transferase (Takara) with 0.25 mM ddNTP (Roche) for 1 hour at 37°C. The fully ligated library was purified by size-selection with agarose-gel electrophoresis (primary library). For RNaseH-digested samples, 100 ng of the primary library was digested using 1xThermoPol reaction buffer, 3.75 units of RNase H and RNase HII (New England Biolabs) overnight (18 hours) at 37°C. Thirty ng of the primary library with or without RNaseH digestion was heat denatured at 100°C for 3 minutes and chilled at 0°C for 5 minutes. The denatured library was incubated with 10 µM of 701ss adaptor and 750 units of T4 DNA ligase (Kapa Biosystems) overnight (12 hours) at 20°C for secondary-adaptor ligation. After three times of library purification with AMPureXP beads (Beckman Coulter), complementary strand of the libraries were synthesized in 25 µl reaction mixture with 16 units of BstPolymerase LF, 1xThermoPol reaction buffer (New England Biolabs), 0.25 mM dNTP and 4µM of TruHTAmp7Rv primer at 65°C for 10 minutes. The size-selected libraries were directly used for paired-end sequencing by MiSeq sequencer (Illumina) to identify location of fragments and 3'-end of nicks generated with or without RNaseH treatment. For PCR-amplified sample, 5 ng of the double-stranded libraries were amplified using 5 µM of primers TruHTAmp5Fw and TruHTAmp701allSRv, using KAPA real time library amplification kit (Kapa Biosystems). Libraries were then purified with AMPureXP beads (target range over 300 bp) and sequenced as PCR-amplified samples.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina MiSeq

Description

Parental yeast strain, YP-0.05% Dextrose culture G1-arrested by alpha factor replicate 1, extract DNA, Covaris shearing, spike-in standard fragments addition, TruSeqHT-compatible adaptor ligation, 3'OH masking by TdT and ddNTP, RNaseH digestion, heat denatured library, second index-adaptor ligation, complementary-strand synthesis, PCR-free sequencing, secondary index fraction, rNMP-detection sample
processed data file: WT_CR_replicate1_Fw.bedgraph
processed data file: WT_CR_replicate1_Rv.bedgraph

Data processing

library strategy: RiSQ-seq
Basecalls performed using Real Time Analysis (RTA) 1.18.42
Background reads and rNMP reads were mapped to EF4.68 assembly using bowtie 1.0.0 with paired-end mapping.
Mapped pair reads (SAM) were converted to fragments and rNMP position (BED) by AWK scripts.
PCR-amplified reads were also converted to rNMP position (BED) by AWK scripts, and adjusted to PCR-free library scale.
Compute read coverage to single-base bin files using bedtools 2.24.0.
rNMP detection efficiency of each sample preparation was estimated from spiked-in standards by linear regression model using R 3.2.1 (lm).
rNMP accumulation level at each 42 base bin was estimated by normalization of total rNMP counts by total background counts with spiked-in standard adjustment.
For nuclear genome, rNMP reads were from PCR-amplified reads adjusted to PCR-free scale.
Genome_build: S. cerevisiae, S288C genome, EF4.68
Supplementary_files_format_and_content: Bedgraph files include rNMP accumlation level, damage per million (dpm); BED file includes biased mapping region with abberant score of rNMP level by mapping bias.

Submission date

Aug 03, 2016

Last update date

May 21, 2021

Contact name

Tetsushi Iida

E-mail(s)

iidatessi@gmail.com

Phone

+81-3-5841-7860

Organization name

The Institute for Quantitative Biosciences (IQB), The University of Tokyo