|
| Status |
Public on May 18, 2021 |
| Title |
2o_plasmid |
| Sample type |
SRA |
| |
|
| Source name |
pc-AT7-Glo1 plasmid with Vex-seq library in configuration 2o
|
| Organism |
synthetic construct |
| Characteristics |
cell line: n/a
sequenced molecule: plasmid DNA
|
| Growth protocol |
Cells were grown to a density of 0.7x10^6 for HepG2 and 1.0x10^6 for K562 and transfected with 5 micrograms of plasmid DNA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total RNA was extracted 48 hours after transfection using Maxwell RSC simplyRNA kit (Promega).
cDNA was synthesized using Superscript III and a reporter-gene specific primer. Illumina primers and indices were added during PCR amplification.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
Amplicon DNA sequencing of final plasmid pool
|
| Data processing |
Library strategy: RT-PCR sequencing (Vex-seq)
UMIs were extracted from read 2 using UMItools (v1.0.0) extract (with --bc-pattern=NNNNNNNNNN) using UMItools
Read pairs were aligned to variant specific references using hisat2 (2.1.0) with parameters -no-softclip --dta-cufflinks.
Duplicate reads were deduplicated using UMItools (v1.0.0) dedup.
Stringtie (v2.0.3) was used to detect novel splice junctions from aligned and deduplicated reads and all samples were merged
Pysam (v0.15.4) was used to extract junction counts from bam files for all stringtie detected junctions.
BWA-MEM (v0.7.17) was used to align plasmid DNA samples.
Samtools and htslb (v1.9) mpileup and bcftools were used to identify the number of novel variant reads and reference reads.
Genome_build: hg19
Supplementary_files_format_and_content: All processed data files are tab separated files. For the plasmid libraries, these contain total counts and counts of variant sequences (1o) as well as the allele frequency. For the Vex-seq libraries these are counts of included reads, excluded reads, percent spliced in, for each variant and identified isoform as well as characteristics about the splice junctions used (Stringtie outputs and pysam parsed junction counts).
|
| |
|
| Submission date |
Jan 13, 2021 |
| Last update date |
May 18, 2021 |
| Contact name |
Scott Adamson |
| E-mail(s) |
adamson@uchc.edu
|
| Organization name |
UConn Health
|
| Department |
Genetics and Genome Sciences
|
| Lab |
Graveley
|
| Street address |
400 Farmington Ave
|
| City |
Farmington |
| State/province |
CT |
| ZIP/Postal code |
06032 |
| Country |
USA |
| |
|
| Platform ID |
GPL27609 |
| Series (1) |
| GSE164752 |
Functional characterization of splicing regulatory elements |
|
| Relations |
| BioSample |
SAMN17307427 |
| SRA |
SRX9843174 |
