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Sample GSM1438838

Query DataSets for GSM1438838

Status

Public on Sep 30, 2015

Title

Alpha_12Y30

Sample type

SRA

Source name

adult brain

Organism

Dinoponera quadriceps

Characteristics

genotype/variation: WT
social status: reproductive

Extracted molecule

polyA RNA

Extraction protocol

Genomic DNA and total RNA were extracted from single brains using the Qiagen All Prep DNA/RNA Mini kit according to the manufacturers’ instructions.
For RNA-seq libraries, 50 to 200 ng total RNA was enriched for mRNA using Dynabeads Oligo(dT)25 from Invitrogen in two subsequent steps of purification with fresh beads. Fragmentation was done by incubation of mRNAs 5min at 94°C in first strand buffer (Invitrogen) and directly followed with cDNA synthesis using SuperScript III kit (Invitrogen). dUTPs were incorporated for second strand synthesis for libraries orientation. cDNA was end repaired, A-tailed and ligated with methylation Adaptor Oligo Kit (Ilumina) using the NEB Next kit according to the manufacturers’ instructions. dUTP excision was done prior to amplification using USER mix (NEB). Libraries were amplified with 16 cycles using 2x Phusion HF buffer (NEB). Size selection and cleaning between steps were performed with the AMPure XP system (Agencourt) to select DNA fragments between 250 and 500 bp. Paired-end libraries were sequenced on either Illumina GAIIx or HiSeq machines.
For BS-seq libraries, synthetic genomes of P. canadensis and D. quadriceps were prepared using REPLI-g Ultra fast Mini kit from QIAGEN and 1 µL of amplification were treated in parallel with the other libraries. Libraries were prepared using 200 to 500 ng genomic DNA. After sonication (Covaris), DNA was end-repaired, A-tailed and ligated with methylation Adaptor Oligo Kit (Ilumina) using the NEB Next kit according to the manufacturers’ instructions. The adaptor-ligated DNA was treated with sodium-bisulfite using the Imprint DNA Modification Kit from Sigma-Aldrich according to the manufacturer’s instructions for the two-step protocol. Bisulfite-treated DNA was amplified using KAPA HiFi Uracil+DNA Polymerase with 15 cycles. Size selection and cleaning between steps were performed with the AMPure XP system (Agencourt) to select DNA fragments between 250 and 500 bp.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Libraries were sequenced on either the Illumina Genome Analyzer IIx or HiSeq platform using the default RTA analysis software.
RNA-Seq reads were aligned using Tophat (v2.0.9) with Bowtie 2 (parameters -g 1).
Bisulfite sequencing reads were trimmed using Trim Galore (v0.3.5) with default parameters (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Reads were aligned and methylation calls extracted using Bismark v0.12.2 (parameters: --bowtie2 --score_min L,0,-0.4).
RNA-Seq data were quantitated using the RNA-Seq pipeline in SeqMonk (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/), counting the log2 transformed number of reads per transcript corrected per million reads (log2RPKM).
Genome_build: Polistes canadensis JPHQ00000000
Genome_build: Dinoponera quadriceps JPHR00000000
Supplementary_files_format_and_content: The genome-wide CX methylation report is tab-delimited, uses 1-based genomic coordinates for every cytosine position in the genome (top and bottom strand) and is in the following format: For Polistes canadensis files: <chromosome> <position> <strand> <count methylated> <count non-methylated> <C-context> <trinucleotide context>; For Dinoponera quadriceps files: <contig> <position> <strand> <count methylated> <count non-methylated> <C-context> <trinucleotide context>
Supplementary_files_format_and_content: Quantitated RNA-Seq data files are tab delimited and contain the following columns: (1) mRNA name; (2) Chromosome; (3) Start; (4) End; (5) Strand; (6) Description; (7-end) log2RPKM Expression Values

Submission date

Jul 17, 2014

Last update date

May 15, 2019

Contact name

Felix Krueger

E-mail(s)

fkrueger@altoslabs.com

Organization name

Altos Labs

Department

Bioinformatics