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Sample GSM1248666

Query DataSets for GSM1248666

Status

Public on Oct 02, 2014

Title

RNA_Ae322_gyne_heads

Sample type

SRA

Source name

gyne heads

Organism

Acromyrmex echinatior

Characteristics

colony id: Ae322
caste: gyne
tissue: heads

Treatment protocol

RNA and DNA samples were collected from three castes: (i) gynes (AeG), (ii) large workers (AeL) and (iii) small workers (AeS). Animals were flash frozen in liquid nitrogen, and the heads separated from the bodies with a pair of forceps.

Growth protocol

Colonies of Acromyrmex echinatior (numbers Ae322, Ae356 and Ae363) were collected in Gamboa, Panama in 2006-2008 and maintained in the laboratory under standard conditions of ca. 25°C and ca. 70% relative humidity where they were supplied with a diet of bramble leaves, rice and pieces of apple.

Extracted molecule

total RNA

Extraction protocol

Heads from 50 gynes, 50 large workers or 200 small workers originating from the same colony were pooled and used for RNA extraction as described previously (Yek et al. 2013). The remaining body parts were likewise pooled per caste and colony and subsequently used for DNA extraction. Genomic DNA was extracted using a Blood & Cell Culture Midi kit (Qiagen) following the instructions enclosed in the kit, with the modification that sample lysis was performed overnight.
For DNA samples, 5 μg genomic DNA per sample was fragmented by sonication with a Covaris S2 system (Covaris, MA) to a mean size of approximately 500 bp, followed by end-repair, 3’-end addition of dA, and adapter ligation. The ligated fragments were size selected at 500 bp on an agarose gel and amplified by 10 cycles of PCR to yield the DNA libraries. All libraries were subjected to 90 bp pair-end sequencing on an Illumina HiSeq 2000 platform.
For RNA samples, strand-specific RNA-Seq libraries were constructed according to Parkhomchuk D, et al (2009). Briefly, 2 μg total RNA per sample was first treated with DNase I (New England BioLabs) to remove the possible contamination of genomic DNA. Then poly (A+) mRNA was purified from the total RNA using the Dynabeads mRNA Purification Kit (Invitrogen), followed by fragmentation using the RNA fragmentation kit (Ambion). Next, the first cDNA strand was synthesized using random hexamer primers and reverse transcriptase (Invitrogen), and second-strand cDNA was synthesized using DNA polymerase I (New England BioLabs) where dUTP was used instead of dTTP. After that, the synthetic double-strand cDNA was end repaired, followed by 3’-end addition of dA, adapter ligation and size selection according to Illumina’s protocols. Finally, the selected products were treated by UNG (Applied Biosystems) and amplified by 15 cycles of PCR to yield the strand-specific RNA-Seq libraries. All the libraries were subjected to 90 bp pair-end sequencing on the Illumina HiSeq 2000 platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

gyne_RNA replicate A

Data processing

library strategy: DNA-Seq for DNA samples and Strand-specific RNA-Seq for RNA samples
RNA and DNA reads were aligned to the genome of Acromyrmex Echinatior (Aech_2.0_scaffolds.fa.gz from http://hymenopteragenome.org/acromyrmex/) using BWA (Version: 0.5.9-r16), allowing 5 mismatches and 0 gap for the 90-bp reads. Only uniquely mapped reads with mapping quality >= 20 and with no suboptimal hits found by BWA were kept for further analysis. Reads resulted from PCR duplications (i.e. read pairs that map to identical genomic locations) were removed except for the one with the highest sequencing quality. Then, we cut the first and last 6-bp of the aligned reads, as it is known that (a) the error rate of Illumina sequencing is relatively higher on both end of a read, especially on the 3’end, (b) some 5’ end mismatches may be introduced by the random hexamer during the first- and second-strand syntheses during RNA library construction and (c) mapping errors around insertions/deletions relative to a reference genome can lead to mismatches occurring towards the beginning and ends of a read. In addition, to improve the accuracy of reads alignment, we used BLAT to re-align all the uniquely mapped reads to the reference genome. As the fundamental algorithmic between BLAT and short read aligners (e.g. BWA) are different, their read mapping results are often different and complementary, especially in handling reads derived from spliced junctions. The reads that could be aligned to positions that are different from BWA alignments with mismatch rates no more than that reported by BWA were removed.
Genome_build: Aech_2.0_scaffolds.fa.gz from http://hymenopteragenome.org/acromyrmex/
Supplementary_files_format_and_content: tab-delimited text files include DNA read coverage and RNA read coverage for each RNA editing site for each sample.

Submission date

Oct 23, 2013

Last update date

May 15, 2019

Contact name

Hong zhi Cao

E-mail(s)

caohongzhi@genomics.cn

Phone

8675525274032

Organization name

BGI-SHENZHEN