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Sample GSM958916

Query DataSets for GSM958916

Status

Public on Jun 03, 2013

Title

Mo17_DNA_rep1

Sample type

SRA

Source name

5-day-old coleoptyle

Organism

Zea mays

Characteristics

tissue: 5-day-coleoptyle
strain: Mo17

Growth protocol

Seeds were grown in the incubator at 25°C in darkness on wet paper towels set in glass Pyrex dish. After 5 days coleoptiles were excised and stored at -80°C.

Extracted molecule

genomic DNA

Extraction protocol

For DNA extraction, coleoptiles were ground with mortar and pestle in LN2, and nuclei were washed and separated from organelles by centrifuging through 30% OptiPrep (Axis-Shield PoC, Oslo, Norway, cat #1114542) solution. For each library, 1-5 μg of genomic DNA was sheared using Covaris S220 Adaptive Focused Acoustics ultra sonicator. Libraries were constructed following standard protocol using the NEBNext DNA Sample Prep Master Mix Set 1 (NEB E6040) and Illumina-compatible paired-end adaptors in which all cytosines were methylated. Each library (50 ng) was treated with sodium bisulfite using the EZ DNA Methylation-GoldTM Kit (Zymo Research D5005) according to the manufacturer’s protocol. The purified library (5-10 ng) was amplified using Expand High Fidelity PLUS PCR system (Roche 03300242001), which is capable of efficiently amplifying uracil-containing templates. PCR reactions (50 μl final volume) containing 200 μM each dNTP, 1 μM each primer, 2.5 mM MgCl2 , and 2.5 U Expand HiFi enzyme were performed according to the manufacturer’s instructions for 18 cycles. Amplified libraries were run on 2% MetaPhor® agarose (Lonza 50108) gel. Fragments of 220-350 bp were excised from gel and purified using the QIAquick PCR Purification kit (Qiagen 28104).DNA concentrations were quantified on a Bioanalyzer (Agilent), diluted to 10 nM and loaded on flow cells to generate clusters. Libraries were sequenced on Illumina GAII or HiSeq2000 machines using the paired-end 50 cycle protocol.

Library strategy

Bisulfite-Seq

Library source

genomic

Library selection

RANDOM

Instrument model

Illumina Genome Analyzer

Data processing

Base call and quality scores were generated using the standard Illumina Pipeline.
BS-seq reads were trimmed for adaptor sequence and low quality base call, then aligned to ZmB73 4a.53 genome assembly using rmap V2.03 with parameters -Q -v -m 4
Then the C/T counts from aligned BS-seq reads for each cytosine in reference genome were saved in CT count file
RNA-seq reads were aligned to ZmB73 4a.53 and calculated FPKM using Tophat v1.3.2/Cufflinks v1.1.0
smRNA-seq reads were aligned to ZmB73 4a.53 genome aeembly using rmap V2.03 with parameters -Q -v -m 2, then the mapped 21/22/24 nt smRNA-seq reads were counted in each 1M bin in whole genome
Genome_build: ZmB73 4a.53
Please note that the bed files for B73 DNA rep 1,2,3 and M017 DNA rep 1,2,3,4 Samples are on the Series record.
Supplementary_files_format_and_content: The CT count files have the first 6 columns the same as bed file, the 7th col is count of C, the 8th col is count of T, the 9th col is the 3 nt in reference genome, the 10th col is the context of cytosine
Supplementary_files_format_and_content: The FPKM tracking file format is described in http://cufflinks.cbcb.umd.edu/manual.html
Supplementary_files_format_and_content: The BED from smRNA-seq data contains the count of mapped smRNA-seq reads in each 1M bin in the whole genome

Submission date

Jul 11, 2012

Last update date

May 15, 2019

Contact name

Zhenyuan Lu

Organization name

Cold Spring Harbor Laboratory

Lab

Doreen Ware

Street address

1 Bungtown Rd