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Sample GSM8008059

Query DataSets for GSM8008059

Status

Public on Apr 01, 2024

Title

A. calliptera, testes, rep4

Sample type

SRA

Source name

Testes

Organism

Astatotilapia calliptera

Characteristics

tissue: Testes
genotype: wild-type

Extracted molecule

total RNA

Extraction protocol

Tissue pieces were transferred to BeadBug tubes prefilled with 0.5 mm Zirconium beads (Merck, #Z763772) and 500-600 µl of TRIzol (Life Technologies, #15596026) was added to the tubes and mixed vigorously. Afterwards, we conducted the homogenisation using a BeadBug microtube homogeniser (Sigma, #Z764140) at approximately 4oC (in cold room). Each sample was homogenised with five BeadBug runs at maximum speed (4000 rpm) for 60 seconds each. Supernatant was then removed into a clean 1.5 mL tube. Centrifuged the lysates again, this time at maximum speed (approximately 21,000 G) for 5 minutes at 4oC. Transferred supernatant into a clean tube without disturbing the pellet and tissue debris. Mixed supernatant thoroughly 1:1 with 100% ethanol, pipetted the mix into a column provided in the Direct-zol RNA Miniprep Plus kit (Zymo Research, #R2072) and followed manufacturer’s instructions, using the recommended in-column DNase I treatment.
Quality control, library preparation (directional, with poly-A enrichment), and mRNA sequencing (Illumina, PE150) was performed by Novogene. A. calliptera brain libraries and P.nyererei gonad libraries were prepared differently with the following protocol. We used 50-250 ng of total RNA for library production with the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB, #E7490), in conjunction with the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB, #E7760) and the NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs, NEB #E6440). Samples were then pooled in equimolar amounts and sequenced on a NovaSeq 6000 system (PE150 on one lane of an S1 Flowcell).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Aca_tes_rep4
rawCounts_mRNA_GenesAndTEs_Malawi_curatedTEAnnotation.txt
NormCounts_mRNA_TEs_Malawi_curatedTEAnnotation.txt
rawCounts_mRNA_GenesAndTEs_Acal.txt
NormCounts_mRNA_TEs_Acal.txt
rawCounts_mRNA_Acal_brain_gonads.txt
lengthScaledTPM_mRNA_Acal_brain_gonads.txt

Data processing

Illumina adapters and reads with low-quality calls were filtered out with Trimmomatic v0.39 (Bolger et al., 2014) using options SLIDINGWINDOW:4:28 MINLEN:36.
We mapped trimmed reads to the genome, using STAR v2.5.4b (Dobin et al., 2013) with options --outTmpDir --readFilesCommand zcat ‑‑genomeDir ‑‑readFilesIn {R1} {R2} --outSAMtype BAM SortedByCoordinate ‑‑outFilterType BySJout --outFilterMultimapNmax 150 ‑‑winAnchorMultimapNmax 150 --alignSJoverhangMin 8 --alignSJDBoverhangMin 3 ‑‑outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.04 --alignIntronMin 20 --alignIntronMax 10000000 --alignMatesGapMax 100000000 --outFileNamePrefix. Reads from Lake Malawi cichlid species (A. calliptera, M. zebra, T. sp. "mauve") were all mapped to the A. calliptera genome fAstCal1.2. A. burtoni, P. nyererei, and O. niloticus reads were mapped to their genomes: AstBur1.0, PunNye1.0, and O_niloticus_UMD_NMBU respectively. All these genomes are available at Ensembl.
The resulting BAM files were used as inputs for TEtranscripts v2.2.1 (Jin et al., 2015) with options -t -c --GTF --TE --stranded reverse ‑‑project --SortByPos.
For A. calliptera samples, gene expression (without TEs) was quantified from trimmed reads using salmon v1.5.1 (Patro et al., 2017), with options --seqBias --gcBias - validateMappings -l A -1 ‑2 -o -i.
DESeq2 (Love et al., 2014) and custom scripts were used to calculate log-normalized and TPM counts, generate plots and conduct statistical tests on an R framework (R Core Team, 2021).
Assembly: fAstCal1.2; AstBur1.0; PunNye1.0; O_niloticus_UMD_NMBU
Supplementary files format and content: rawCounts_mRNA_GenesAndTEs_Acal.txt: Tab-delimited file includes raw counts for genes and TEs in each A. calliptera sample. Output of TEtranscripts.
Supplementary files format and content: NormCounts_mRNA_TEs_Acal.txt: Tab-delimited file includes DESeq2 normalized counts for TEs in each A. calliptera sample.
Supplementary files format and content: rawCounts_mRNA_GenesAndTEs_Malawi_curatedTEAnnotation.txt: Tab-delimited file includes raw counts for genes and TEs in each Lake Malawi cichlid sample. Output of TEtranscripts, using a curated TE annotation.
Supplementary files format and content: NormCounts_mRNA_TEs_Malawi_curatedTEAnnotation.txt: Tab-delimited file includes DESeq2 normalized counts for TEs in each Lake Malawi cichlid sample. Using a curated TE annotation.
Supplementary files format and content: rawCounts_mRNA_Acal_brain_gonads.txt: Tab-delimited file includes raw counts in each A. calliptera sample. From Salmon output.
Supplementary files format and content: lengthScaledTPM_mRNA_Acal_brain_gonads.txt: Tab-delimited file includes length scaled TPM counts in each A. calliptera sample. From Salmon output.
Supplementary files format and content: rawCounts_mRNA_GenesAndTEs_Abur.txt: Tab-delimited file includes raw counts for genes and TEs in each A. burtoni sample. Output of TEtranscripts.
Supplementary files format and content: NormCounts_mRNA_TEs_Abur.txt: Tab-delimited file includes DESeq2 normalized counts for TEs in each A. burtoni sample.
Supplementary files format and content: rawCounts_mRNA_GenesAndTEs_Pnye.txt: Tab-delimited file includes raw counts for genes and TEs in each P. nyererei sample. Output of TEtranscripts.
Supplementary files format and content: NormCounts_mRNA_TEs_Pnye.txt: Tab-delimited file includes DESeq2 normalized counts for TEs in each P. nyererei sample.
Supplementary files format and content: rawCounts_mRNA_GenesAndTEs_Onil.txt: Tab-delimited file includes raw counts for genes and TEs in each O. niloticus sample. Output of TEtranscripts.
Supplementary files format and content: NormCounts_mRNA_TEs_Onil.txt: Tab-delimited file includes DESeq2 normalized counts for TEs in each O. niloticus sample.

Submission date

Jan 09, 2024

Last update date

Apr 01, 2024

Contact name

Miguel Vasconcelos Almeida

E-mail(s)

[email protected], [email protected]

Organization name

University of Cambridge

Department

Department of Biochemistry