|
| Status |
Public on Dec 04, 2021 |
| Title |
long day IP rep1 |
| Sample type |
SRA |
| |
|
| Source name |
heads
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Oregon-R
developmental stage: Adult
treatment: long day(16:8)
antibody: AGOI
|
| Treatment protocol |
One group was exposed to lond days (LD16:8) and a second gropu was exposed to short days (LD8:16) during the development and until tissue (heads) collection.
|
| Growth protocol |
~40 eggs of OR-R flies maintained at 25ºC LD 12:12 were collected and placed either in long (LD 16:8) or short (LD 8:16) day (18ºC). The flies developed in these conditions for 20 days and the heads of emerging females (3-4 days old) were collected at ZT3.5 (3.5 hours after light on) in liquid nitrogen
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Two-hundred µl (volume) of frozen fly heads where homogenized in 600 µl of lysis buffer (30 mM HEPES KOH pH 7.4, 100 mM KAcetate, 2 mM MgAcetate, 5% glycerol, 0.1% Triton X-100, 1 mM EGTA, 5 mM DTT, 0.4 U/ul RNAse OUT [Invitrogen], 25 mM EDTA). The lysate was incubated in ice for 10min and centrifuge at 14000rpm for 15min at 4ºC. 100 µl of the supernatant was isolate as input while the rest (~450 µl) was used for the immunoprecipitation. 15 µl of anti-AGO-1 antibody 38 was added to the supernatant and incubated at 4ºC O/N with rotation. 100 µl of protein-G plus beads slurry (MERCK, IP04-1.5ML) was washed trice with 250 µl of lysis buffer and then added to the lysate and incubated at 4ºC for 4 hours. The beads were recovered by a 2min centrifugation at 8000rcf and washed 5 times in 500 µl lysis buffer at 4ºC with rotation. Finally, the RNA in the immunoprecipitation and input was isolated with TRIzol (Thermo-Fisher Scientific) following manufacture instructions.
The RNA-seq library preparation and sequencing was carried by Glasgow polyomics (http://www.polyomics.gla.ac.uk/) using an IlluminaNextseq500 platform. Two independent libraries (pair-end) for long day and one for short day (input and IP) were generated.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
LD1-RIP
|
| Data processing |
Adapter sequences and poor quality readings were trimmed from the RNA-seq data with trimmomatic (version 0.32) 39. The quality of the resulting trimmed libraries was checked with fastQC (0.11.2)
For the alignment we used the software STAR (version 2.5.2b) 41 with these options --runMode alignReads -genomeDir genomeIndex (this contain the index for the reference genome) --sjdbGTFfile genes.gtf (from NCBI_build5.41) as gene annotation reference --sjdbOverhang 100 --outSAMstrandField IntronMotif --outFilterIntronMotifs RemoveNoncanonical.
Samtools (version 1.3.2) 42 was used to convert sam to bam files to sort them and index them
To quantify the expression of transcripts (genes) of the aligned RNA we initially used cufflinks (vrsion 2.2.1), cuffmerge and cuffquant 43-46 with these options: -u --library-type fr-unstranded -g genes.gtf (from NCBI_build5.41) as gene annotation reference -N -b genome.fa (from NCBI_build5.41/Bowtie2Index) as reference genome -M Mask_sequences.fa (including mitochondrial and ribosomal RNA as well as polyA and polyC sequences).
The expression was expressed in Fragments Per Kilobase of transcript per Million fragments mapped (FPKM). Finally, to calculate enriched transcripts in IP (Input vs IP) and differences between samples (long vs short day) we used cuffdiff with a geometric method of normalization
Genome_build: D. melanogaster transcriptome (NCBI_build5.41) downloaded from the illumina igenome website
Supplementary_files_format_and_content: expression data for all individual samples and Ratio RIP/IN per treatment (LD and SD)
Supplementary_files_format_and_content: expression data for all samples (gropued by treatment_input_RIP), including all pairwise comparison and associated qvalues
|
| |
|
| Submission date |
Dec 02, 2021 |
| Last update date |
Dec 04, 2021 |
| Contact name |
Mirko Pegoraro |
| E-mail(s) |
[email protected]
|
| Phone |
+4401512312175
|
| Organization name |
Liverpool John Moores University
|
| Department |
School of Natural Science and Psychology
|
| Lab |
Evolutionary Epigenetics Lab
|
| Street address |
Byrom Street
|
| City |
Liverpool |
| State/province |
Liverpool |
| ZIP/Postal code |
L33AF |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL19132 |
| Series (1) |
| GSE189992 |
RNA_Seq of AGOI_IP long (16:8), short (8:16) day heads |
|
| Relations |
| BioSample |
SAMN23564276 |
| SRA |
SRX13283352 |
