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Sample GSM2045652

Query DataSets for GSM2045652

Status

Public on Nov 06, 2017

Title

Sample_23620_Hyp

Sample type

SRA

Source name

Hypothalamus

Organism

Zonotrichia albicollis

Characteristics

Sex: M
tissue: Hypothalamus
morph: TS
Stage: I
age: SY

Extracted molecule

total RNA

Extraction protocol

Brains were quickly removed and flash frozen on dry ice. Brains were cryosectioned at 300um thickness and regions of interest (HYP and MeA) regions were micro dissectied from frozen setions using a 1mm punch tool. A total of 4 punches were collected from each bird and region and were pooled for RNA extraction. RNA was extracted using the Qiagen Allprep DNA/RNA micro kit with modification, as follows. Punches were homogenized in RLT plus + β-mercaptoethanol using a pellet mixer. An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) pH 7.5 was added to each sample. Samples were further homogenized by passing them through a Qiashredder column (Qiagen). Phases were separated by spinning for 15 min at 4°C, and the aqueous phase was collected and further processed according to the manufacturer’s protocol.
Libraries were prepped using Illumina TruSeq RNA Sample Preparation Kit v2. Libraries were constructed using 250 ng total RNA.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 1000

Description

NCBI_plus_Zal2m_minqual0_htseq-count_table.txt
Male Hyp 10W 9T NCBI_2m_MinQual0 VST_13605.csv

Data processing

The reference genome (Zonotrichia albicollis 1.0.1, GCA_000385455.1), was generated from a bird homozygous for the ZAL2 haplotype. In order to eliminate potential mapping bias against reads that originated from the ZAL2m chromosome, we created an enhanced reference genome that contained sequences representing both ZAL2 and ZAL2m haplotypes within the inversion. To create this enhanced version of the white-throated sparrow genome, we first performed a ~30x whole-genome shotgun sequencing of a ‘superwhite’ bird homozygous for the ZAL2m chromosome (Horton et al., 2013, SRA accession pending). Novoalign (novocraft.com, version 3.00.05) was used to map the reads to the reference genome using ‘-r RANDOM –I PE 510,150 -0 SAM’ option. To identify single nucleotide substitutions (SNPs) between the ZAL2 and ZAL2m chromosomes, Samtools (Li et al., 2009) was used to call SNPs between the reference genome and the superwhite genome. High-quality (genotype quality score = 99) homozygous non-reference SNPs were then used to generate alternative contigs representing ZAL2m sequences within the inversion (Supplemental File 1). To create these ZAL2m contigs, we first identified NCBI genome contigs that mapped to ZAL2 via orthology with zebra finch chromosome 3 (Davis et al., 2011; Romanov et al., 2011) based on sequence similarity and gene annotations. We then created ZAL2 m versions of the contigs that mapped within the inversion using the GATK tool FastaAlternativeReferenceMaker (McKenna, et al., 2010). We also generated assemblies of rRNA and the mitochondrial genome derived from these whole-genome shotgun sequences (Supplemental Files 2-3). Our final enhanced reference genome was composed of the NCBI reference genome (Zonotrichia albicollis 1.0.1) plus ZAL2m versions of contigs located within the inversion, rRNAs, and a mitochondrial genome.
Sequenced reads were mapped to the enhanced reference genome using the STAR aligner (Dobin et al, 2013), with the parameter outFilterMultimapNmax=2. Reads that did map within the inversion to both the reference and alternative contigs were then filtered to retain just the primary mapping location. All other dual mappings were excluded. Retained mappings to the alternate contigs were relabeled to conform to the unmodified reference contig names.
Reads were assigned to genes using htseq-count (Anders et al., 2015) based on NCBI RefSeq annotations.
Samples were divided into two datsets based on morph. Within each dataset, genes with 0 reads in more than half the samples were removed. Reads were then transformed using the 'varianceStabilizingTransformation' function in DeSeq2 implemented in R.
Supplementary_files_format_and_content: Tab delineated text file containing gene read counts for each sample and comma delineated text files containing variance stabilizing transformed read counts for each sample.

Submission date

Jan 25, 2016

Last update date

May 15, 2019

Contact name

Donna L Maney

Organization name

Emory University

Street address

1510 Clifton Rd NE Rm 2006

City

Atlanta