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Sample GSM5251916

Query DataSets for GSM5251916

Status

Public on May 02, 2024

Title

VZ_A19_E30 replicate 1

Sample type

SRA

Source name

Ferret brain (microdissected regions of ferret cortex) Embryonic day 30

Organism

Mustela putorius furo

Characteristics

age/developmental stage: Embryonic day 30
treatment: Caudal Sulcus (A19)
tissue: microsdissected VZ layer

Treatment protocol

None

Growth protocol

Ferret embryos were obtained by cesarean section of timed?pregnant females upon deep anesthesia with sodium pentobarbital and then perfused transcardially with 4% paraformaldehyde (PFA); postnatal ferrets were deeply anesthetized with sodium pentobarbital prior to transcardiac perfusion with PFA. After perfusion, the brains were extracted, cryoprotected, frozen, and sectioned.

Extracted molecule

total RNA

Extraction protocol

Cells were cross-linked in medium containing 1% formaldehyde for 10 min at room temperature, neutralized with 0.125M glycine, scraped off and rinsed twice with cold 10 ml 1X PBS. Cells were pelleted by centrifugation for 7 min at 4°C at 600g. Pellets were resuspended in 10 mL of Buffer L1 (50mM Hepes KOH, pH 7.5, 140mM NaCl, 1mM EDTA pH 8.0, 10% glycerol, 5% NP-40, 0.25% Triton-X 100) and incubated at 4°C for 10 min. This was followed by centrifugation for 5 min at 4°C at 1300g. The pellet was again resuspended in 10 ml of Buffer L2 (200mM NaCl, 1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 10mM Tris pH 8.0) and incubated at room temperature for 10 min, followed by centrifugation for 5 min at 4°C at 1300g. The pellet was then resuspended in 900 µl Buffer L3 (1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 10mM Tris pH 8.0, 100mM NaCl, 0.1% Na-deoxycholate, 0.17mM N-Lauroyl sarcosine) containing protease inhibitors and incubated at 4°C for overnight following sonication using Bioruptor plus (Diagenode). After clearing cellular debris by spinning at 14,000 g for 10 min at 4°C DNA 60 µg of chromatin were incubated at 4 °C overnight with respective antibodies after 1h of preclearing. The mixture was then incubated with 40 µl protein A-Sepharose beads preblocked with tRNA and BSA for 3 h at 4°C. Beads were washed twice with 1 ml buffer L3 and once with 1 ml DOC buffer (10 mM Tris (pH 8.0), 0.25 M LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA), and bound chromatin was eluted in 1% SDS/0.1 M NaHCO3. This followed treatment with RNase A (0.2mg/ml) for 30 min at 37°C and then with Proteinase K (50 µg/ml) for 2.5 h at 55°C. The crosslinking was reversed at 65°C overnight with gentle shaking. DNA was purified by phenol-chloroform extraction followed by ethanol precipitation and recovered in 40 µl TE buffer.
1. RNAseq libraries were prepared for sequencing using Truseq stranded illumina protocol; 2. ChIP-seq libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

RNA-seq (single end)
VZ_A19_E30_R1
RNAseq_normalizedCount.csv

Data processing

The RNA-sequencing output was in FASTQ format. After a quality check using FASTQC, the output was aligned to the Ferret genome MusPutFur1.0 (GCA_000215625.1) with Ensembl’s annotations using TopHat2.1.1. Only uniquely mapped reads were retained for further analysis. SAMTOOLS v0.1.19 (Li et al, 2009) was used to convert the BAM output to SAM format and to sort the BAM file. The read counts per gene were calculated using the HTSeq program, (Anders et al, 2015). The DESeq2 package was used to generate normalized read counts and for differential gene expression analysis.
ChIP-sequencing files in fastq format were subjected to quality control using FASTQC. Bowtie2 was used with local parameter for soft clipping of reads and align the reads to Ferret genome MusPutFur1.0 (GCA_000215625.1). Aligned sam files were sorted, indexed, and converted to the bam format using SAMTOOLS v0.1.19. Peak calling was performed using MACS2 and taking Input as a control. Peaks were merged from all samples as well as replicates to retain all the regions of interest for differential occupancy. qCount from QuasR was used to count reads from all samples in the merged peak regions.
Genome_build: MusPutFur1.0 (GCA_000215625.1)
Supplementary_files_format_and_content: 1. Normalised read Counts for all RNA-seq samples; 2. Read counts in merged peaks regions for ChIP-seq

Submission date

Apr 18, 2021

Last update date

May 02, 2024

Contact name

Aditi Singh

E-mail(s)

neuro.aditis@gmail.com

Organization name

Queen's University Belfast

Department

The Wellcome-Wolfson Institute for Experimental Medicine, SMDBS