|
| Status |
Public on Nov 04, 2021 |
| Title |
20E injection - rep1 |
| Sample type |
SRA |
| |
|
| Source name |
central brain
|
| Organism |
Harpegnathos saltator |
| Characteristics |
tissue: central brain
caste: worker
treatment: 20E
|
| Treatment protocol |
Each ant was injected with a calibrated glass capillary needle directly into the head, right below the antennas with 100 ng of 20E or 100 ng of JH3 dissolved in 5 % glucose. 10 hours after the injections, the central brain was dissected in neurobasal medium, and processed for RNA extraction.
|
| Growth protocol |
Animals were housed in a plaster nest in a clean, temperature (25°C) and humidity (50%) controlled ant facility on a 12-hour light/dark cycle and fed three times per week with live crickets.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Harpegnathos central brains were dissected from single individuals, snap-frozen and then homogenized in TRIzol (Thermo Fisher Scientific, MA). RNA was purified and its quality visualized on agarose-formaldehyde gels. Typical yields amounted to 0.6 µg total RNA per central brain.
For library preparation, polyA+ RNA was isolated from 600 ng total RNA using Dynabeads Oligo(dT)25 (Thermo Fisher) and constructed into strand-specific libraries using the dUTP method (Parkhomchuk et al., 2009). UTP-marked cDNA was end-repaired using end-repair mix (Enzymatics, MA), tailed with deoxyadenine using Klenow exo- (Enzymatics), and ligated to custom dual- indexed adapters with T4 DNA ligase (Enzymatics). Libraries were size-selected with SPRIselect beads (Beckman Coulter, CA) and quantified by qPCR after amplification.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
internal reference:
190514.HYFMWBGX9.b0101
20E_10h_d10_Inj1_s1
|
| Data processing |
Reads were demultiplexed using bcl2fastq2 (Illumina) with the options “--mask-short-adapter-reads 20 --minimum-trimmed-read-length 20 --no-lane-splitting --barcode-mismatches 0”. Reads were aligned to the H. saltator v8.5 assembly (Shields et al., 2018) using STAR (Dobin et al., 2013). STAR alignments were performed in two passes, with the first using the options “--outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 7 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.07 --alignIntronMin 20 --alignIntronMax 100000 --alignMatesGapMax 250000”, and the second using the options “--outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.04 --alignIntronMin 20 --alignIntronMax 500000 --alignMatesGapMax 500000 --sjdbFileChrStartEnd [SJ_files]” where “[SJ_files]” corresponds to the splice junctions produced from all first pass runs.
Gene-level read counts were produced using featureCounts (Liao et al., 2014) with the options “-O -M --fraction -s 2 –p”, against a custom adapted NCBI Harpegnathos annotation (see following paragraph) using gene-level values (not individual mRNAs). Resulting counts were rounded to integers prior to importing into R for DESeq2 analysis in order to account for fractional count values associated with fractional counting of multi-mapping reads.
Genome_build: v8.5
Supplementary_files_format_and_content: matrix of per-sample normalized FPKM values for all protein-coding genes in the Hsalt NCBI annotation (release 102)
|
| |
|
| Submission date |
Nov 10, 2020 |
| Last update date |
Feb 04, 2022 |
| Contact name |
Roberto Bonasio |
| Organization name |
University of Pennsylvania
|
| Department |
Cell and Developmental Biology
|
| Lab |
Bonasio
|
| Street address |
3400 Civic Center Blvd - SCTR 9-111
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL29395 |
| Series (2) |
| GSE161203 |
RNA-seq in Harpegnathos central brains after hormone injection |
| GSE161207 |
Functional genomic analyses on the role of Kr-h1 in the 20E/JH3 pathways in Harpegnathos ants |
|
| Relations |
| BioSample |
SAMN16727762 |
| SRA |
SRX9470615 |
