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Sample GSM3402482

Query DataSets for GSM3402482

Status

Public on May 29, 2019

Title

WT pollen RNA-seq rep4

Sample type

SRA

Source name

WT pollen

Organism

Arabidopsis thaliana

Characteristics

cell type: whole pollen
genotype: WT

Growth protocol

plants were grown in LD condition (16h lights/8h darkness) at 22-24 degrees

Extracted molecule

total RNA

Extraction protocol

follow the instruction of Mirvana miRNA isolation kit
follow the instructions of ScriptSeq v2 RNA-seq lib prep kit

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

mikado-permissive.superloci.gff
TAIR10_gene_plus_mikado_TE_transcripts.gff
This sample was used only for TE transcript identification and not the RPKM calculations.

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).
RNA-Seq: TE transcript annotation was created using four biological replicates of pollen RNA-seq data. Tophat2, Hisat, and STAR were used to align the RNA-seq reads to the TAIR10 genome
Transcripts were assembled using CLASS2, StringTie, and Cufflinks, respectively.
Assembled transcripts were selected by Mikado using default options except the BLAST and Transdecoder steps were disabled. As a result, 21381 transcripts (called superloci) were identified and included in the output file called mikado-permissive.superloci.gff.
The list of superloci was refined by selecting those overlapping with TAIR10 TE annotation.
TE-like genes were eliminated from the refined list if their CG methylation is less than 0.7 in rosette leaves. This gave rise to an annotation of TE transcripts.
The annotation of TE transcripts was combined with TAIR10 gene annotation for Kallisto analysis which gives rise to the output files named abundance.tsv.
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 26, 2018

Last update date

May 29, 2019

Contact name

Xiaoqi Feng

E-mail(s)

xiaoqi.feng@jic.ac.uk

Organization name

John Innes Centre

Department

Cell and Developmental Biology