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| Status |
Public on Jan 05, 2024 |
| Title |
20A_plus_single_Rhino_ChIP_IP, rep2 |
| Sample type |
SRA |
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| Source name |
adult fly ovaries gDNA (ChIP)
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| Organism |
Drosophila melanogaster |
| Characteristics |
developmental stage: adult
tissue: ovaries
reporter: 20A cluster single reporter insertion plus orientation
chip antibody: Rhino (From Julius Brennecke lab)
fraction: gDNA (ChIP)
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| Treatment protocol |
Flies were fed with yeast for 2-3 days prior to dissection
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| Growth protocol |
Fly stocks were maintained at 24C on standard diet
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total smRNA from ovary was isolated with Ribozol reagent and do size selection from 19nt to 30nt. For ChIP, ovaries were fixed using Paraformaldehyde (PFA) at a final concentration of 1.8% in PBS for 10min at RT. Samples were quenched by 125mM glycine for 5 min at RT and washed 3 times in PBS. Ovaries were afterwards slightly dounced in Farnham Buffer (5mM HEPES pH8.0; 85mM KCl; 0.5% NP-40/Igepal; Protease Inhibitor Cocktail; NaF 10mM; Na3VO4 0.2mM) followed by strong douncing in RIPA Buffer (20mM Tris pH7.4; 150mM NaCl; 1% NP-40/Igepal; 0.5% Sodium Deoxycholate; 0.35% SDS; Protease Inhibitor Cocktail; NaF 10mM; Na3VO4 0.2mM). Lysates were sonicated using Bioruptor (Diagenode, 30 cycles of 30sec ON, 30sec OFF) then add IP diluted buffer (20mM Tris pH7.4; 150mM NaCl; Protease Inhibitor Cocktail; NaF 10mM; Na3VO4 0.2mM). Supernatants were pre-cleared for 2h at 4°C using Dynabeads Protein G (Invitrogen) beads. 5% of samples were saved for inputs. The rest of the lysates were incubated with antibodies for 2h to overnight at 4°C, then added BSA and DNA blocked Dynabeads Protein G beads and incubated for 3h at 4°C beads The beads were washed 5 times at 4°C with LiCl IP wash buffer (10mM Tris pH 7.4; 500mM LiCL; 1%NP40/Igepal; 1% Sodium Deoxycholate) and once with TE. Protein was digested with proteinase K for 3hrs at 55°C and reverse-crosslinked by incubation at 65°C overnight. DNA was extracted by phenol:chloroform standard protocol.
smRNA-seq libraries were made by using NEBNext® Small RNA Library Prep Set for Illumina®. Libraries were sequenced on the Illumina HiSeq 2000 platform. ChIP-seq library construction was carried out using the NEBNext ChIP-Seq Library Prep Master Mix Set (E6240) with minor modifications. After adaptor ligation and PCR amplification, size selections were done with AMPure beads to select the 200bp-600bp size window. Libraries were sequenced on the Illumina HiSeq 2000 platform.
smRNA-seq follow NEBNext® Small RNA Library Prep Set for Illumina®, ChIP-seq follow NEBNext ChIP-Seq Library Prep Master Mix Set
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
ChIP-seq reads were trimmed by Trimmomatic (version 0.33) and cutadapt (version 1.15) and filter out those shorter than 50 bp after trimming
The first 50bp of reads were mapped to dm3 genome and vectors sequence respectively, using Bowtie (version 1.0.1, parameters: -v 2 -k 1 -m 1 -t --best -y --strata)
Aligned reads were then used to generate piled-up RPM signals and enrichment profiles by our customized scripts and deepTools
Read counts over equal-sized bins were calculated using deepTools2 and BEDOPS, and plus, minus strand of genome regions and vectors were separate, figures were made by Matlab
smRNA-seq reads were trimmed by Cutadapt to rRNA and filter out those shorter than 15 bp after trimming
Select reads size 21-22bp (siRNA) and 23-29bp (piRNA) and 21-30bp (small RNA)
mapped to dm6 genome and extracted regions 20A cluster(20A) and 42AB cluster(42A), mapped to vector maps, Ubi-GFP(UBIG), non-RMCE Ubi-GFP(originalUBIG), double-stranded GFP reporter(UBIGasG) (parameters: -v 0 -a -m 1 -t --best --strata), and plus, minus strand of genome regions and vectors were separate.
smRNA profilings were generated by Matlab after mapping
Genome_build: dm3(ChIP-seq) and dm6(smRNA-seq)
Supplementary_files_format_and_content: bg4
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| Submission date |
Jan 05, 2022 |
| Last update date |
Jan 05, 2024 |
| Contact name |
Yicheng Luo |
| E-mail(s) |
[email protected]
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| Phone |
5167762261
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| Organization name |
Calfironia Institute of Technology
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| Department |
Division of Biology and Biological Engineering
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| Street address |
1200 E California Blvd
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| City |
Pasadena |
| State/province |
CA |
| ZIP/Postal code |
91125 |
| Country |
USA |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE193091 |
Maternal inherited siRNA initiate piRNA cluster formation |
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| Relations |
| BioSample |
SAMN24656658 |
| SRA |
SRX13617590 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5773096_20A_plus_single_Rhino_ChIP_IP_2.dm3.50mer.originalUBIG.vectoronly.dup.10.bg4.chopped.bg4.txt.gz |
3.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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