|
| Status |
Public on Nov 12, 2020 |
| Title |
Het_12hr_Sample_3 |
| Sample type |
SRA |
| |
|
| Source name |
Tcf3-/- Mouse ESC + Human B cell line
|
| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
tissue: Tcf3-/- Mouse ESC + Human B cell line
strain: 129/Sv
|
| Treatment protocol |
Tc3-/- mouse embryonic stem cells were labeled with Vybrant DiD and Human B lymphocytes were labeled with Vybrant DiO. Both labeled cells were mixed and treated withpolyethylene glycol (PEG) at 37 deg for a maximum period of 2.5 mins. Cells were then resuspended and washed with DMEM and plated in DMEM media supplemented with 10% foetal bovine serum and mouse LIF. Finally at specific timepoints the cells labeled with both dyes were sorted by flow cytometry for RNA extraction.
|
| Growth protocol |
Tcf3-/- mouse embryonic stem cells were cultured in DMEM media supplemented with 10% foetal bovineserum and murine LIF. Human B lymphocytes were cultured in RPMI media supplemented with 20% foetal bovine serum
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cells were processed using the Qiagen RNA extraction kit for isolating the RNA
RNA Samples isolated from the heterokaryons were further processed to generate sequencing libraries using a Truseq RNA library Prep Kit.
The libraries were then analyzed on an Illumina HiSeq 2000 sequencer using 100 bp paired-end sequencing.
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
12 hour Fused Heterokaryons
|
| Data processing |
Alignment of the FASTQ Files were performed using bowtie2 (version 2.2.6) aligner. Alignment was done using using --no-mixed --no-discordant flags to eliminate ambiguous read pairs and only counting reads where both pairs were mapped. PE data was directly converted to sorted BAM format using the samtools API.
BAM files were converted into EntrezGene count files using the summarizeOverlaps function in the GenomicAlignments package (v 1.8.4) using "union" mode, dropping reads mapped to multiple features. Reads were counted using the MM10 and HG19 genomes downloaded from the UCSD Genome Browser database.
Multi-mapping reads were corrected in 2 steps. First, reads that mapped to both genomes but aligned within a gene boundary for only one genome were assigned to aligned gene boundary and genome. Second, reads that mapped gene boundaries for both human and mouse genomes were evenly split between both genes. For example if a read mapped to Human Gene A that already had 4 reads mapped to it, and Mouse Gene B that already had 6 reads assigned to it, then Gene A would receive 0.4 reads, and Gene B would receive 0.6 reads. Additional details for the correctly algorithm can be found in the associated publication. Uploaded count files have been aligned, mapped, and corrected using these methods.
Genome_build: Human (HG19) and Mouse (MM10) genomes were downloaded from the Illumina igenome database (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) for indexing and aligment with bowtie2. For generation of count files, MM10 and HG19 genomes were downloaded from the USCD Genome Browser database (http://hgdownload.soe.ucsc.edu/downloads.html)
Supplementary_files_format_and_content: Count Files contain the corrected count values for Human and Mouse genes.
|
| |
|
| Submission date |
May 09, 2018 |
| Last update date |
Nov 12, 2020 |
| Contact name |
Karthik Arumugam |
| Organization name |
Centre for Genomic Regulation
|
| Street address |
C/Dr.Aiguader, 88
|
| City |
Barcelona |
| State/province |
Barcelona |
| ZIP/Postal code |
08025 |
| Country |
Spain |
| |
|
| Platform ID |
GPL16512 |
| Series (2) |
| GSE114238 |
RNA sequencing analyses of fused heterokaryons between mouse embryonic stem cells and human B lymphocytes collected at early timepoints after fusion |
| GSE114240 |
The Master Regulator protein BAZ2B can reprogram human hematopoietic lineage-committed progenitors into a multipotent state |
|
| Relations |
| BioSample |
SAMN09098996 |
| SRA |
SRX4059512 |
