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Sample GSM5560089

Query DataSets for GSM5560089

Status

Public on Sep 27, 2022

Title

RAB5A overexpression Replicate 3 SAMMY-seq fraction S3

Sample type

SRA

Source name

MCF10DCIS.com

Organism

Homo sapiens

Characteristics

cell line: MCF10DCIS.com
treatment: RAB5A overexpression
fraction: Chromatin fraction S3

Treatment protocol

MCF10.DCIS.com were infected with pSLIK-hygro-RAB5B, pSLIK-neo-RAB5C, pSLIK-neo-EV (empty vector, CTR) or pSLIK-neo-RAB5A lentiviruses and selected with the appropriate antibiotic to obtain stable inducible cell lines. Constitutive expression of mCherry H2B was achieved by retroviruses infection of MCF10DCIS.com cells with pBABE-puro-mCherry-H2B vector. pLL5.0 E-Cadherin shRNA/mEcad-GFP vector was a gift from Alpha S. Yap (Division of Molecular Cell Biology, Institute for Molecular Bioscience, The University of Queensland, Australia). pTRIP-CMV-GFP-FLAG-cGAS GFP vector was from Addgene (plasmid# 86675). pTRIP-SFFV-EGFP-NLS vector was from Addgene (plasmid# 86677). Transfections were performed using either calcium phosphate or FuGENE HD Transfection Reagent (Promega, Cat# E2311), according to the manufacturer’s instructions.

Growth protocol

MCF10.DCIS.com cells were provided by J. F. Marshall (Barts Cancer Institute, Queen Mary University of London, UK) and maintained in DMEM/F12 (Biowest) supplemented with 5% horse serum (Life Technologies), 2 mM L-Glutamine (EuroClone), 0.5 mg/ml Hydrocortisone (Sigma-Aldrich), 10 μg/ml Human insulin (Sigma-Aldrich) and 20 ng/ml EGF (Peprotech).

Extracted molecule

genomic DNA

Extraction protocol

Three distinct biological replicas of control and RAB5A expressing MCF10.DCIS.com monolayers were processed for chromatin fractionation. 3 million cells were washed in PBS 1X, and extracted in 600 ml of cytoskeleton buffer (CSK: 10 mM PIPES pH 6,8; 100 mM NaCl; 1 mM EGTA; 300 mM Sucrose; 3 mM MgCl2; 1X protease Inhibitors by Roche Diagnostics; 1 mM PMSF) supplemented with 1 mM DTT and 0,5% Triton X-100. After 10 min on wheel at 4°C the cytoskeletal structure was separated from soluble proteins by centrifugation at 900g for 3’ at 4°C, and the supernatant was labeled as S1 fraction. The pellets were resuspended with 600 ml of cytoskeleton buffer, put 10 min on wheel at 4°C followed by centrifugation at 900g for 3’ at 4°C. Chromatin was solubilized by DNA digestion with 25U of RNase–free DNase (Turbo DNAse; Invitrogen AM2238) in 100 ml of CSK buffer for 60 min at 37°C. To stop digestion, ammonium sulphate was added in CSK buffer to a final concentration of 250 mM and, after 5’ in ice samples were pelleted at 900g for 3 min at 4°C and the supernatant was labeled as S2 fraction. The pellets were resuspended with 200 ml of CSK buffer, put 10 min on wheel at 4°C followed by centrifugation at 3000g for 3 min at 4°C. The pellet was further extracted with 100 l of CSK buffer with 2M NaCl for 5 min at 4°C, centrifuged at 2300g 3 min at 4°C and the supernatant was labeled as S3 fraction. This treatment removed the majority of histones from chromatin. The pellets were washed twice with 200 ml of CSK buffer with 2M NaCl, put 10 min on wheel at 4°C followed by centrifugation at 3000g for 3 min at 4°C., The pellets were solubilized in 100 ml of 8M urea buffer for 10 min at room temperature to remove any remaining protein component by applying highly denaturing conditions. This fraction was labeled as S4. DNA was extracted from S2, S3 and S4 fractions. All fractions were quantified and analyzed by SDS-PAGE and immunoblotting. Anti-tubulin alpha (Sigma T5168, mouse 1:10000), H3 (Abcam ab1791, rabbit 1:4000), Beta-Actin (Santa-Cruz sc1616, rabbit 1:4000), were used as primary antibodies. HRP-conjugated secondary antibodies were revealed with the ECL chemiluminescence kit (Thermo Fisher Scientific). After incubation 90 min at 37°C with 6 l of RNAse cocktail (Invitrogen AM2286) followed by 150 min at 55°C with 40 mg of Proteinase K (Invitrogen, AM2548), DNA was extracted by standard phenol/chloroform extraction, precipitated and resuspended in 15μl milliQ H2O. The S2 fraction was further purified with PCR DNA Purification Kit (Qiagen, 28106). After Qubit HS DNA quantification then samples were evaluated by capillary electrophoresis (Agilent 2100 Bioanalyzer) and then sonicated with a Covaris M220 Focused-ultrasonicator using screw cap microTUBEs with the parameters: water bath 20°C, peak power 30.0, duty factor 20.0, cycle/burst 50, duration: 125 seconds for S2 and S3 fractions, 150 seconds for S4 fraction. The DNA profiles were checked again by capillary electrophoresis (Agilent 2100 Bioanalyzer).
For fractions S2, S3 and S4 obtained from chromatin fractionation procedure, at least 2.5 ng DNA were used to generate an indexed library (Kapa HyperPrep kit; Roche KK8504, KK8727). Indexed DNA libraries were purified (AmpureXP, Beckman, A63881), quantitated (Qubit dsDNA HS Assay, Q32851), checked for size distribution on Agilent Bioanalyzer 2100 (DNA HS kit, Agilent, 5067-4626) and normalized for pooling. 1% PhiX control was added to the sequencing pool, to serve as a positive run control. Sequencing was performed in SR mode (1x75nt) on an Illumina NextSeq550 platform, generating at least 30 million SR reads per sample.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

NextSeq 550

Description

Chromatin fraction S3 as per the original SAMMY-seq protocol (Sebestyén et al, Nature Communication 2020)

Data processing

Library strategy: SAMMY-seq
Alignment: Sequencing reads were trimmed and adapters removed by using Trimmomatic (v0.39) using the following parameters for SAMMY-seq: 2 for seed_mismatch, 30 for palindrome_threshold, 10 for simple_threshold, 3 for leading, 3 for trailing and 4:15 for sliding window and sequence minimum length threshold of 35. As clip file has been used the trimmomatic provided dataset “TruSeq3-SE.fa” (for single end). All reads were cropped to 75 bp reads length (if longer) by setting the crop option of Trimmomatic (v0.39) to 75. After trimming, the reads were aligned using BWA (v0.7.17-r1188) etting –k parameter as 2 and using as reference genome the UCSC hg38 one (only canonical chromosomes have been taken into consideration). The alignment duplicates have been marked with Picard (v2.22) (http://broadinstitute.github.io/picard/) MarkDuplicates option. And then filtered using Samtools (v1.9), in addition we filtered all the reads with mapping quality lower than 1, unmapped and read fails platform/vendor quality checks (-F 1540 -q 1). Each sequencing lane has been analysed separately up to this point and then merged.
Genomic tracks: The comparison between sequencing reads enrichment in ChIP-seq was performed using the SPP (v1.16.0) R (v3.5.2) library. The reads have been imported from the (previously filtered) bam files using the “read.bam.tags” function, then they were filtered using “remove.local.tag.anomalies” and finally the normalized log2 reads density ratio was computed using the function “get.smoothed.enrichment.mle” setting “tag.shift = 0” and “background.density.scaling = TRUE” to exclude enriched regions from the calculation of normalization scaling factor.
Genome_build: UCSC hg38 one (only canonical chromosomes have been taken into consideration)
Supplementary_files_format_and_content: BigWig (.bw) files contains genomic tracks information.
Supplementary_files_format_and_content: Bed (.bed) files contains peak information (ChIP over INPUT enriched regions)

Submission date

Sep 06, 2021

Last update date

Sep 27, 2022

Contact name

Giorgio Scita

E-mail(s)

giorgio.scita@ifom.eu

Organization name

IFOM, The AIRC Institute of Molecular Oncology

Street address

via Adamello, 16