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Sample GSM5221275

Query DataSets for GSM5221275

Status

Public on Aug 11, 2021

Title

NPC H2B-Dam repl1

Sample type

SRA

Source name

Neural progenitors cells

Organism

Mus musculus

Characteristics

cell line: NPC derived in vitro from E14
antibody: none
treatment: 100ng/mL doxycycline + 1uM Shield1 for 15h

Treatment protocol

H2B-Dam and H2B-Dam-NoLS cell lines on the 8th day of differention in NPC were harvested with 10X trypsin to get single cells from the embryoid bodies. 1 x 106 cells Neural progenitor cells were seeded in 6-well plates coated with 0.1% gelatin and treated with 100ng/ml Doxycycline and 1µM Shield1 (Clontech Takara) for 15h.

Growth protocol

Neural progenitor cells were generated from ESCs, according to a previously established protocol (Bibel et al., 2004) using a suspension-based embryoid bodies formation (Bacteriological Petri Dishes, Bio-one with vents, Greiner). The neural differentiation media (DMEM, 10% fetal calf serum, 1× MEM NEAA, Pen/Strep, β-mercaptoethanol, Sodium Pyruvate) was filtered through 0.22 μm filters and stored at 4°C. During the 8-day differentiation procedure, media was exchanged every 2 days. In the last 4 days of differentiation, the media was supplemented with 2 μM retinoic acid to generate neural precursors that are Pax-6-positive radial glial cells.

Extracted molecule

genomic DNA

Extraction protocol

DNA extracted using Quick-DNA Miniprep Plus kit (Zymo Research).
DamID seq was performed on H2B-Dam and H2B-Dam-NoLS Doxycycline and Shield1-induced samples adapting the previously described protocol to work with Next Generation sequencing (Vogel et al., 2007). Briefly, 500ng of gDNA previously extracted with Quick-DNA Miniprep Plus kit (Zymo Research) was digested for 4h at 37°C with DpnI (New England Biolabs) to cut methylated GATC sites. After heat inactivation for 20 minutes at 80°C, DamID adaptors (Vogel et al. 2007) were blunt-end ligated overnight at 16°C and then heat inactivated at 65°C for 10 minutes. In order to cut unmethylated GATC sequences, DNA was digested for 3h at 37°C with DpnII (New England Biolabs) and heat inactivated at 65°C for 20 minutes. Adaptor-ligated fragments were amplified using the Advantage® GC 2Polymerase mix (Clontech Takara) (primer described in Table MM, Vogel et al., 2007). DamID libraries were purified using Agencourt AMPure XP beads (Beckam Coulter). Since the size of fragments produced exceeded the one fitting the sequencing machine, the libraries were fragmented using the ds Fragmentase (New England Biolabs) to enrich the concentration of the libraries below 500bp and, afterwards, the libraries were purified again using the Agencourt AMPure XP beads (Beckam Coulter). The quantity and quality of the isolated DNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies, California, USA). The Nugen Ovation Ultra Low Library Systems (Nugen, Inc, California, USA) to prepare the libraries for Illumina sequencing. Briefly, Nucleolar-DamID samples (1 ng) were end-repaired and polyadenylated. Then, Illumina compatible adapters, containing the index for multiplexing, were ligated. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Bioanalyzer 2100 (Agilent, Waldbronn, Germany). The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. The TruSeq SR Cluster Kit v4-cBot-HS (Illumina, Inc, California, USA) was used for cluster generation using 8 pM of pooled normalized libraries on the cBOT. Using the TruSeq SBS Kit v4-HS (Illumina, Inc, California, USA) the sequencing was performed as single end 150bp reads using the Illumina NovaSeq 6000.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

NPC_NADs_Nucleolar-DamID_score.csv

Data processing

Library strategy: DamID-seq
Fastaq files were aligned to the mm10 genome assembly using Bowtie2 (version 2.3.4.3) with default parameters
The bam files were analyzed using the damidseq pipeline script from the Brand group (http://owenjm.github.io/damidseq_pipeline) (Marshall and Brand, 2015). The pipeline bins the mapped reads into GATC-fragments according to GATC-sites indicated by a gff file for the GRCm38 mouse genome (already provided by the authors of the pipeline on the website above) and normalizes reads against the Dam control, in our case the H2B-Dam sample. The pipeline gives a bedgraph file with the log2 ratio of the m6A between H2B-Dam-NoLS and the H2B-Dam only. The bedgraph files were processed with the find_peaks software associated with the pipeline (https://github.com/owenjm/find_peaks) adjusting the values of the FDR and the minimum quantile (FDR <0.01 and min_quant 0.70). Only the significative peaks common to both replicates were considered as NADs for further analysis. The intersection of identified NADs and previously published lamina associated domains (LADs, Peric-Hupkes et al., 2010) results in the classification of NADonly, NAD/LAD, and LAD.
for ChIP seq data,read counts were computed and normalized using “bamCoverage” from deepTools (version 3.2.1) using a bin size of 50bp.
for RNA SEQ, reads were aligned to the reference genome (ensembl version 82) with Subread (i.e. subjunc, version 1.4.6-p4; 50) allowing up to 16 alignments per read (options: –trim5 10 –trim3 15 -n 20 -m 5 -B 16 -H –allJunctions). Count tables were generated with Rcount 51 with an allocation distance of 100 bp for calculating the weights of the reads with multiple alignments, considering the strand information, and a minimal number of 5 hits. For each gene, exon coverage was calculated using a custom pipeline and then normalized in reads per kilobase per million (RPKM) (Mortazavi et al., 2008), the method of quantifying gene expression from RNA sequencing data by normalizing for total read length and the number of sequencing reads.
Genome_build: mm10

Submission date

Mar 31, 2021

Last update date

Aug 11, 2021

Contact name

Raffaella Santoro

E-mail(s)

raffaella.santoro@dmmd.uzh.ch

Phone

+41 44 635 54 75

Organization name

University of Zurich