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| Status |
Public on Dec 14, 2023 |
| Title |
mEC RNAiSvb rep2 |
| Sample type |
SRA |
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| Source name |
mEC RNAiSvb
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: GAL4/GAL80TS system allows spatial and temporal control of UAS induction. UASs were activated in ISCs for 6 days before midgut dissection
tissue: Posterior midgut (R3 to R5)
age: 9 days (6 days of induction)
Sex: female
genotype: MyoIA-Gal4, UAS-GFP/+; tubGal80TS/UAS-SvbRNAi
cell type: Enterocytes (mature)
treatment: 3 days old female flies were transferred to 29°C for 6 days, to induce UASs expression.
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| Treatment protocol |
Batches of >20 virgin females are CO2-anaethesized and put on ice. From then, all solutions are used cold and samples kept on ice whenever possible. Their midgut are dissected in 1XPBS and transferred to a clean PBS dish. Malpighian tubules and hindgut are removed and only the posterior part of the midgut is kept (R3, R4, R5). PBS was replaced by PBS1X-EDTA 1mM. Posterior midguts were transferred on a glass slide in a droplet of PBS, and shrinked into pieces using a razorblade. Samples were transferred into a fresh tube and spun 3- 5minutes at 1000rpm 4°C to pellet the tissue and remove all supernatant except 50μL. 200uL TrypLE 10X (8X final with 0.2mM EDTA) was added for enzymatic digestion. Then the guts were incubated at 37°C and rocked vigorously for 10 minutes. Digestions were stopped with 750μL SSM (Serum Supplemented Medium = S2 Cell culture medium). Trituration was made by pipetting up and down using low binding, flame-rounded narrow tips (only 5 times with narrow tip, no use of the very narrow tip).
Transfer tissue onto a PBT-coated 70μM nylon cell strainer in SSM. Transfer the strainer into clean SSM, then transfer into PBS-EGTA. The aim is to have dissociated individual cells (comprising progenitors) flow through the strainer while patches of multiple ECs stay on the strainer. After an additional PBS-EGTA wash, turn the strainer bottom up on a petri dish and rinse abundantly with PBS-EDTA (5mL) then 2mL SSM to detach the EC patches. Make sure your EC samples look healthy under fluorescent microscope. Transfer samples into tubes, spin (700 rcf at 4°C for >10minutes) to pellet the cells in 150uL, add 150uL Tissue and Cell Lysis solution + 1ug Proteinase K [50ug/uL], vortex and proceed with RNA extraction.
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| Growth protocol |
MyoIA-Gal4, UAS-GFP; tubGal80TS/SM66B flies were crossed to the following strains: Canton-S (BL#64349), UAS-SvbRNAi (VDRC# 41584), UAS-SvbACT::GFP (al Hayek et al., 2021), UAS-Svb3Kmut::GFP (al Hayek et al., 2021). Adult progeny (only virgin females) was aged 3 days at room temperature, then transferred at 29°C for 6 days prior to midgut dissection.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA were purified from each cell sample using MasterPure RNA purification kit (Epicentre), following manufacturer’s recommendation. Sorted cells were incubated at 65°C with rocking for 15 minutes. Samples were put on ice then total nucleic acid were precipitated. Contaminating DNA was removed using DNase, then nucleic acids were precipitated again. RNAs were finally resuspended in 10uL TE and 1uL RiboGuard (RNases inhibitor) and stored at -20°C.
Construction of RNA banks and sequencing was made at the Montpellier GenomiX Platform (MGX). RNA banks were obtained using the Ovation SoLo RNAseq System kit (Nugen). Bank validation was done with the quantification of the complementary DNA with the Standard Sensitivity NGS kit on Fragment Analyzer and with qPCR (ROCHE Light Cycler 480). 100nt single-reads sequencing was performed on NovaSeq 6000 (Illumina) with NovaSeq Reagent Kits (100cycles).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
D2_m_RNAi
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| Data processing |
Quality of sequencing was measured with fastQC (v0.11.5)
Sequenced reads were mapped on Drosophila genome (r6.13 Flybase) with STAR (v 2.5.2b default parameters)
Reads were counted with HTseq-count (v 0.6.0, parameter -t gene -r pos - i gene_symbol)
Statistical analysis were performed with edgeR using the negative binomial generalized log-linear model to the read counts ( gmLRT), the TMM normalization and decideTest were used to determine the differentially expressed genes.
Clustering analysis was performed on the matrix of the reads count average centrered and reduced for each condition previously filtered for the gene expression (difference of read > 50 between condition.
Two steps of clustering were done. The hierarchical ascendant clustering was performed with R function hclust() with the Spearman distance and the Ward.D2 option.
The k-means clustering was done with R function kmeans() with default options and k = 5. The kmeans clustering was repeated 15 times ( 5 times with predefined centroid and 10 with random centroids). The predefined centroids are genes of differents cluster defined by the hierarchical clustering.
Different k-means clusters obtained by each repetition were compared with cluster_similarity (Clusteval v 0.1). Clustering is similar if the score eaqual 1.
Assembly: dm6
Supplementary files format and content: bigwig smooth
Supplementary files format and content: Matrix table with normalized counts, logFC, Pvalue, LogCPM, status for expressed genes
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| Submission date |
Dec 08, 2022 |
| Last update date |
Dec 14, 2023 |
| Contact name |
Cedric Polesello |
| Organization name |
Centre de Biologie Intégrative de Toulouse
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| Street address |
118 Route de Narbonne
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| City |
Toulouse |
| ZIP/Postal code |
310362 |
| Country |
France |
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| Platform ID |
GPL25244 |
| Series (2) |
| GSE220560 |
Determination of Shavenbaby target genes in mature Enterocytes (mECs) |
| GSE220561 |
Determination of Shavenbaby target genes in Intestinal Stem Cells differenciation |
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| Relations |
| BioSample |
SAMN32301624 |
| SRA |
SRX18541469 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6806902_mEC_RNAiSvb_2_RPKM_smooth.bw |
33.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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