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Sample GSM4790438

Query DataSets for GSM4790438

Status

Public on Jul 01, 2021

Title

OSC - Piwi-KD - LamDam_rep2

Sample type

SRA

Source name

Ovarian Somatic Cells

Organism

Drosophila melanogaster

Characteristics

cell line: Ovarian Somatic Cells
treatment: Piwi-KD

Treatment protocol

For the transfection of siRNAs into OSCs, 200 pmol siRNA duplex was nucleofected into 3.0 × 10^6 cells using the Cell Line 96-well Nucleofector Kit SF (Lonza) and program DG150 of the 96-well Shuttle Device (Lonza). The siRNAs were transfected twice for 4-day KD, and once for 2-day KD. Co-transfection of siRNA and plasmid DNA was performed using the Cell Line Nucleofector Kit V (Lonza) and program T-029 of the Nucleofector II Device (Lonza).

Growth protocol

Ovarian somatic cells (OSCs) was established in the previous studies (Niki et al., 2006; Saito et al., 2009). OSCs were cultured in Shield and Sang M3 Insect Medium (Caisson Laboratories) supplemented with 10% fly extract, 10% fetal bovine serum, 0.6 mg/ml glutathione, and 10 μg/ml insulin.

Extracted molecule

genomic DNA

Extraction protocol

Hi-C: Hi-C using OSCs were performed as described previously (Ulianov et al., 2016) with some minor modifications. OSCs were fixed with 1% formaldehyde for 10min, and stored at -80 degrees before usage. 5 x 10^5 cells were lysed and chromatin digested using 30G needle. Chromatin digestion was performed using HindIII restriction enzyme. Ends were filled with biotin-14-dCTP followed by blunt-end ligation. Proteins were reverse crosslinked form the cells, and DNA purification was performed. Biotin was removed from un-ligated ends and DNA was sonicated using covaris S220 (covaris). Sonicated DNA was used for biotin pull down by streptavidin conjugated to Dynabeads Streptavidin C1 beads (Invitrogen). Pulled-down DNA was used as the template to construct DNA library.
DamID-seq: Dam-ID-seq was performed as described previously (Pickersgill et al., 2006) with some modifications to analyze methylated sequences using deep-sequencing rather than microarray. DamID vectors were transfected into OSCs using Nucleofector, one day before cell harvest. pNDamMyc plasmid (van Steensel and Henikoff, 2000) was a kind gift from Dr. Bas van Steensel. Lamin was cloned into pNDamMyc vector as described previously (Pickersgill et al., 2006). Genome DNA precipitation, DpnI digestion, adapter ligation, DpnII digestion, PCR, and PCR cleanup was performed as described within “Van Steensel Lab version 28/08/2006” protocol. Obtained DNA was used as the template to prepare deep sequencing library.
ChIP-seq: ChIP was performed as described previously (Iwasaki et al., 2016). Briefly, OSCs (3×107) were fixed, lysed, and their nuclei were isolated using truChIP Chromatin Shearing Kits (Covaris), in accordance with the manufacturer’s instructions. DNA was sonicated to ~300 bases using Bioruptor (Cosmobio), and then diluted with ChIP buffer. DNA–protein complexes were incubated with antibody on Dynabeads-Protein G for 4 h, at 4 degrees. Beads were washed with ChIP buffer, high-salt lysis buffer, and wash buffer followed by TE. After reversing cross-linking for 12–16 h at 65 degrees, samples were treated with RNase for 30 min at 37 degrees and Proteinase K for 60 min at 55 degrees. DNA was then recovered.
DNA library was constructed using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB), in accordance with the manufacturer’s instructions.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

HiSeq X Ten

Description

siPiwi_rep2.LamDam-vs-Dam.gatc.bedgraph

Data processing

Library strategy: DamID-seq
Hi-C: HiCExplorer v3.3 software (Ramirez et al., 2018) was used for the calculation of HiC matrix, and GENOVA v1.0 was used for visualizing the results. Two replicates were performed per knockdown, and hicCorrelate was used to calculate correlation between replicate samples. Due to the high correlation between replicates, samples were combined by using hicSumMatrices for the further analysis. Bins of too low or high coverage were removed and the HiC matrix was iteratively corrected by ICE using hicCorrectMatrix. HiC matrix was further normalized between different knockdown samples using hicNormalize. TADs were defined using hicFindTADs function and matrixes were converted into ginteractions file using hicConvertFormat.
DamID-seq: Adapters were removed using cutadapt, and reads shorter than 50nt after adapter removal was discarded. damidseq_pipeline (Marshall and Brand, 2015) was used to map the reads to Drosophila melanogaster (dm3) genome, followed by normalization and background reduction. We used q-value cutoff 0 (to include multi-mapped reads), and fragment size of 300. Due to the high correlation of replicate samples, two replicate reads were merged for the further analysis. After mapping of DamID-seq reads to the genome, log2 ratio of LamDam/Dam was calculated and bedgraph file was generated using damidseq_pipeline.
ChIP-seq: For ChIP-seq analysis, adapters were removed from the reads using Cutadapt, and subsequent reads shorter than 50 nt were discarded. ChIP-seq reads were mapped to the Drosophila genome (dm3) using Bowtie 2.2.9 (Langmead and Salzberg, 2012) with the default parameters. MACS 1.4.2 (Zhang et al., 2008) was used for peak calling and generation of wig files.
Genome_build: dm3
Supplementary_files_format_and_content: Hi-C: ginteractions file (tsv format) generated using HiCExplorer v3.3 (Ramirez et al., 2018)
Supplementary_files_format_and_content: DamID-seq: bedgraph file generated using damidseq_pipeline v1.4.5 (Marshall and Brand, 2015)
Supplementary_files_format_and_content: ChIP-seq: wig file generated using MACS v1.4.2 (Zhang, 2008)

Submission date

Sep 16, 2020

Last update date

Jul 03, 2021

Contact name

Yuka W. Iwasaki

E-mail(s)

iwasaki@keio.jp

Organization name

Keio University School of Medicine

Department

Department of Molecular Biology