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Sample GSM5773037 Query DataSets for GSM5773037
Status Public on Jan 05, 2024
Title 20A_minus_single_smRNA, rep1
Sample type SRA
 
Source name adult fly ovaries total small RNA
Organism Drosophila melanogaster
Characteristics developmental stage: adult
tissue: ovaries
reporter: 20A cluster single reporter insertion minus orientation
fraction: total small RNA
Treatment protocol Flies were fed with yeast for 2-3 days prior to dissection
Growth protocol Fly stocks were maintained at 24C on standard diet
Extracted molecule total RNA
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
 
Library strategy ncRNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model Illumina HiSeq 2000
 
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
 
Submission date Jan 05, 2022
Last update date Jan 05, 2024
Contact name Yicheng Luo
E-mail(s) [email protected]
Phone 5167762261
Organization name Calfironia Institute of Technology
Department Division of Biology and Biological Engineering
Street address 1200 E California Blvd
City Pasadena
State/province CA
ZIP/Postal code 91125
Country USA
 
Platform ID GPL13304
Series (1)
GSE193091 Maternal inherited siRNA initiate piRNA cluster formation
Relations
BioSample SAMN24656717
SRA SRX13617555

Supplementary file Size Download File type/resource
GSM5773037_20A_minus_single_smRNA_1.dm6.23_29mer.unique.dup.bam.1000.20A.Minus.bg4.chopped.bg4.txt.gz 347 b (ftp)(http) TXT
GSM5773037_20A_minus_single_smRNA_1.dm6.23_29mer.unique.dup.bam.1000.20A.Plus.bg4.chopped.bg4.txt.gz 386 b (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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