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Sample GSM2341391

Query DataSets for GSM2341391

Status

Public on Jan 07, 2017

Title

FB3

Sample type

SRA

Source name

Red blood cell

Organism

Geospiza fortis

Characteristics

tissue: Red blood cell
developmental stage: Adult

Treatment protocol

Samples from different cell types, species, and collection locations were compared.

Growth protocol

Red blood cell and sperm samples were collected from wild finches

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA from finch red blood cells (erythrocytes) was prepared using the Qiagen DNAeasy Blood and Tissue Kit (Qiagen, Valencia CA). The manufacturer’s instructions for nucleated blood samples were followed, but in the final DNA elution step H2O was used instead of the buffer provided in the kit. Genomic DNA from finch sperm was prepared as follows: collected sperm suspension was adjusted to 100 μl with 1 x Phosphate Buffered Saline (PBS) then 820 μl DNA extraction buffer (50mM Tris pH8, 10mM EDTA pH8, 0.5% SDS) and 80 μl 0.1 M dithiothreitol (DTT) were added and the sample was incubated at 65°C for 15 minutes. Next, 80 μl Proteinase K (20 mg/ml) were added and the sample was incubated on a rotator at 55°C for 2 hours. After incubation, 300 μl of protein precipitation solution (Promega, A795A) were added, then the sample was mixed and incubated on ice for 15 minutes, then spun at 4°C at 13,000 rpm for 20 minutes. The supernatant was transferred to a fresh tube, then precipitated over night with the same volume of 100% isopropanol and 2 μl glycoblue at -20°C. The sample was then centrifuged and the pellet washed with 75% ethanol, then air-dried and re-suspended in 100 μl H2O. DNA concentration was measured using a Nanodrop Spectrophotometer (Thermo Fisher).
The MeDIP pools were used to create libraries for next generation sequencing (NGS) at the University of Nevada, Reno Genomics Core Laboratory using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®, starting at step 1.4 of the manufacturer’s protocol to generate double stranded DNA. After this step the manufacturer’s protocol was followed.

Library strategy

MeDIP-Seq

Library source

genomic

Library selection

5-methylcytidine antibody

Instrument model

Illumina HiSeq 2500

Description

allForRBC.results.csv.gz

Data processing

Basic read quality was verified using summaries produced by FastQC
The reads for each sample for both CNV and DMR analyses were mapped to the zebra finch (Taenopygia guttata) genome using Bowtie2
The mapped read files were then converted to sorted BAM files using SAMtools
The MEDIPS R package was used to calculate differential coverage between sample groups. To identify DMR, the reference genome was broken into 100 bp windows. The edgeR Pvalue was used to determine the relative difference between the two sample groups for each genomic window. Windows with an edgeR P value less than 1e-3 were considered DMR. The DMR edges were extended until no genomic window with an edgeR P value less than 0.1 remained within 1000 bp of the DMR.
The DMR that included at least two windows with an edgeR P value < 1e-3 were then selected for further analysis and annotated.
Genome_build: taeGut3.2.4
Supplementary_files_format_and_content: The results of the edgeR analysis in CSV format. This includes raw read counts for each genomic window as well as the calculated p-value and other summary values.

Submission date

Oct 11, 2016

Last update date

May 15, 2019

Contact name

Michael K Skinner

E-mail(s)

[email protected]

Organization name

WSU

Department

SBS