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Sample GSM2306313

Query DataSets for GSM2306313

Status

Public on Dec 12, 2017

Title

WT_meiocyte_RNASeq_Rep1

Sample type

SRA

Source name

WT_meiocyte_RNASeq

Organism

Arabidopsis thaliana

Characteristics

ecotype background: Col-0
genotype/variation: wild type
age: 5-6 week
tissue/cell type: male meiocyte

Growth protocol

A. thaliana plants of Col-0 ecotype were grown under 16h light/ 8h dark in a growth chamber.

Extracted molecule

total RNA

Extraction protocol

Stage 9 flower buds were collected and gently squeezed between a glass slide and coverslip. The released meiocytes (of meiotic prophase I) were examined carefully under a microscope. Clean meiocytes free from any somatic cell debris were transferred to a new slide using capillary glass pipettes, washed by 1 x PBS buffer 3 times, and frozen in liquid nitrogen. Sperm cells and vegetative cell nuclei were isolated by Fluorescence-activated cell sorting (FACS)
Single-end bisulfite sequencing libraries for Illumina sequencing were constructed using the Ovation Ultralow Methyl-Seq Library Systems (Nugen, #0336) and EpiTect Fast Bisulfite Conversion (Qiagen, #59802) kits according to the kit protocols, except the incorporation of two rounds of bisulfite conversion.Strand-specific RNA sequencing libraries were prepared using ScriptSeq v2 RNA-Seq (Illumina, #SSV21106) Library Preparation and Ovation RNA-Seq Systems for Model Organisms Arabidopsis (Nugen, #0351) kits

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Bisulfite-Seq: We used Perl scripts to convert all the Cs in the reads (and in the scaffolds) to Ts, and aligned the converted reads to the converted reference scaffold, allowing up to two mismatches per read. 76 base reads were divided into the first 45 and the last 31 bases, 100 base reads and 150 base reads were divided in half. Each half of the read was aligned independently using bowtie, allowing up to two mismatches. The coordinates of the two halves were subsequently correlated; the second half was discarded if it did not match the first.
Single_C: We used Perl scripts to recover the original sequence of each mapped read and, for each C (on either strand), count the number of times it was sequenced as a C or a T.
50bp_window: We used a Perl script to calculate fractional methylation (#C/(#C+#T)) within a 50 bp sliding window for each sequence context (CG, CHG, CHH).
RNASeq: Tophat-2.0.10 followed by Cufflinks-2.2.1 were used to highlight differentially expressed genes between samples.
Genome_build: TAIR10
Supplementary_files_format_and_content: All files are in GFF format. Files contain fractional methylation data either for individual cytosines (single-c) or in 50 bp windows.

Submission date

Sep 08, 2016

Last update date

May 15, 2019

Contact name

Xiaoqi Feng

E-mail(s)

xiaoqi.feng@jic.ac.uk

Organization name

John Innes Centre

Department

Cell and Developmental Biology