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Sample GSM4194591

Query DataSets for GSM4194591

Status

Public on Aug 07, 2020

Title

EB14_loxp_gDNA_es_eb4

Sample type

SRA

Source name

mESC (Mouse embryoid body at day 0)

Organism

Mus musculus

Characteristics

cell type: Embryoid body
media: Serum-LIF
differentiation day: Day 14
treatment: None

Treatment protocol

To perturb DNA methylation, we treated EBs with 5-azacytidine or DMSO (100 nM) and replenished them with every media change throughout all days of differentiation.

Growth protocol

Mouse v6.5 ESCs were cultured with feeder cells (mouse embryonic fibroblasts (MEF)) in knockout DMEM media supplemented with 15% FBS, and 0.01% Leukemia inhibitory factor (LIF). The media was replenished every day and cells were split every two days.
For differentiation, we detached and dissociated ESCs to single cells. MEFs were further removed by incubating single cell suspensions on a 100mm tissue culture dish for 40 minutes at 37°C. We seeded 1000 cells per well micro-well plate (STEMCELL Technologies) in EB media (mESC media without LIF). For inducible barcoding experiments, we seeded FACS sorted RFP+/GFP-/BFPmid cells. After 4 days of EB differentiation in the micro-well plate, we collected EBs and transferred them to 100mm petri dishes and pursued spontaneous differentiation for another 10 days. EB media was changed every day (micro-well plate) or every other day (petri dish) during EB differentiation.

Extracted molecule

genomic DNA

Extraction protocol

mESC and EBs were dissociated by trypsin to single cells and sorted into 384 well plates. After freeze and thaw for twice, we performed a reverse transcription and second strand synthesis, then pooled samples and ran IVT. For DNA extraction (30%), before pooling, we added unique primers (DNA adapter) involving identical cell barcode with RNA-adapter, targeting dual-barcode region of genomic DNA to each well, and then amplified the region by PCR. For RNA extraction (70%), after 2nd reverse transcription, we amplified RNA fragments by Illumina adapter.
DNA libraries were prepared for sequencing using standard Nanopore protocols.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

MinION

Description

EB_day14_loxp_gDNA_es_eb4
DNA-Seq
384 cells
processed data file: EB_recording_dual_barcode.txt

Data processing

Sequencing RNA libraries, passed through general quality filter such as cluster density, total yield, and per-cycle base quality, were then split by library barcodes using bcl2fastq.
R2 reads were demultiplexed into separate files based on the cell barcode from R1 and mapped to a mouse reference genome (mm9) by Bowtie2. After eliminating reads sharing the same UMI by htseq-count, an accurate molecule count for each gene was generated and converted to the number of UMIs into transcript counts through binomial statistics (CEL-seq2).
Nanopore sequencing reads were converted to fastq files by Guppy (v2.3.5) and then demultiplexed to the each cell based on its matched cell barcode sequence with the highest mapping score by minimap2 with default parameters.
To detect tandem-loxP, different loxP flanking sequences were mapped to the demultiplexed nanopore sequence (minimap2) then assembled into a full tandem-loxP sequence based on their position in the nanopore sequence. Due to the sequencing error, we selected the top 1 combination with additional filter: 1) over 30 reads, 2) outlier from others, 3) full length with upstream and downstream sequence.
To detect the UCI, we first mapped the upstream and downstream of the UCI sequence to nanopore reads by minimap2 then extracted the sequence between M13F and WPRE_M13R_HSV. Due to the sequencing error, we selected the top 1 sequence with additional filter: 1) over 30 reads, 2) outlier from others, 3) represented by the sequence from UCI plasmid.
Genome_build: mm9 (MGSCv37)
Supplementary_files_format_and_content: Tab-delimited text files include UMI count values for each Sample.

Submission date

Nov 26, 2019

Last update date

Aug 07, 2020

Contact name

Bradley E Bernstein

E-mail(s)

[email protected]

Organization name

Massachusetts General Hospital

Department

Pathology