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Sample GSM4287582

Query DataSets for GSM4287582

Status

Public on Jun 16, 2021

Title

P106_RNAseq

Sample type

SRA

Source name

Brain

Organism

Polistes canadensis

Characteristics

tissue: Brain
experiment: Control

Extracted molecule

polyA RNA

Extraction protocol

Heads were cut off and immediately placed in RNA later solution (Ambion) and kept at -20°C until brain dissection.
Total RNAwas extracted from single brains using theQIAGENAll PrepDNA/RNAMini Kit according to the manufacturer’s instructions.
50 to 200 ng of 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 for 5 min at 94 °C in the First-Strand Buffer (Invitrogen), and directly followed by cDNA synthesis using a SuperScript III Kit (Invitrogen) according to the manu- facturer’s instructions. dUTPs were incorporated for second-strand synthesis for library orientation except for samples P188 to P212. cDNA was end-repaired, A-tailed, and ligated with a methylated Adaptor Oligo Kit (Illumina) using the NEB Next Kit (New England Biolabs) according to the manufacturer’s instructions. dUTP excision was done before amplification using USER mix (New England Biolabs). Libraries were amplified with 16 cycles using 2× Phusion HF buffer (New England Biolabs). Size selection and cleaning between steps were performed with the AMPure XP system (Agencourt) to select DNA fragments between 250 bp and 500 bp. Paired-end libraries were sequenced on Illumina HiSeq system.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Libraries were sequenced on the Illumina HiSeq platform using the default RTA analysis software.
RNA-Seq data data were trimmed with Trim Galore (v0.4.1, default parameters) and mapped to the Polistes canadensis genome assembly using TopHat v2.0.12 as previously described in Patalano et al. PNAS, 2015 . Strand specific quantification was performed using RNA-seq pipeline in Seqmonk software Version 1.39.0 (www.bioinformatics.babraham.ac.uk/projects/seqmonk/). Gene expression levels were normalized in terms of reads per million (reads per kilobase per million, RPKM). To normalize across nests a scaling factor was calculated using the DEseq2 package in R (Allnest_normSizeFactor.txt) log transform with only orientated samples (ALLRNAseqlogand features).
Genome_build: Polistes canadensis GCF_001313835.1
RNA-seq reports: This file tab-delimited is the strand specific quantification performed using RNA-seq pipeline in Seqmonk version 1.39.0 (www.bioinformatics.babraham.ac.uk/projects/seqmonk/). Gene expression levels were normalized in terms of reads per million (reads per kilobase per million, RPKM). To normalize across nests a scaling factor was calculated using the DEseq2 package in R (Allnests_normSizeFactor.txt) and log transformed for samples P188 to P212 (All_RNAseq_log_and features.txt). Lines indicated individual wasp, column indicated genes.

Submission date

Jan 28, 2020

Last update date

Jun 16, 2021

Contact name

Felix Krueger

E-mail(s)

[email protected]

Organization name

Altos Labs

Department

Bioinformatics