|
| Status |
Public on Nov 04, 2021 |
| Title |
20E -> JH3 - 30 min - rep 3 |
| Sample type |
SRA |
| |
|
| Source name |
primary neuronal cultures
|
| Organism |
Harpegnathos saltator |
| Characteristics |
tissue: primary neuronal cultures
treatment: JH3
culture medium: 20E
timepoint: 30min
|
| Treatment protocol |
After 5 days in JH3 or 20E, the medium was replaced with one containing 2 µM of the opposite hormone (JH3 for 20E cultures or 20E for JH3 cultures) for different lenghts of time or DMSO for 30 minutes as a control.
|
| Growth protocol |
Single-cell suspensions from central brains were plated on poly-D-lysin-coated plates in complete growing media (4 : 6, neurobasal : DMEM medium with 10 mM HEPES, 1x non-essential amino acids, 1x B27 supplement, 1x insulin-transferrin-selenium, 240 nM progesterone and 5 % (v/v) honeybee pupal extract). Cultures were further supplemented with either 2 µM 20E or 2 µM JH3. After four days this medium was replaced with one lacking the honeybee pupal extract.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were lysed 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 |
neu.20E.to.JH3.30min.rep3
internal reference:
191211.HHFVFBGXC.ND68
JH3_30m_bg_20E_s73
|
| 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) |
| GSE161204 |
RNA-seq in primary Harpegnathos neuronal cultures stimulated with 20E or JH3 |
| GSE161207 |
Functional genomic analyses on the role of Kr-h1 in the 20E/JH3 pathways in Harpegnathos ants |
|
| Relations |
| BioSample |
SAMN16727709 |
| SRA |
SRX9470918 |
