|
| Status |
Public on Nov 26, 2019 |
| Title |
Experimental cDNA replicate 2 for MPRA2 |
| Sample type |
SRA |
| |
|
| Source name |
synthetic
|
| Organisms |
Pan troglodytes; Homo sapiens |
| Characteristics |
cell type: H9-derived Neural Stem Cells
passage: 14
|
| Treatment protocol |
MPRA libraries were transfected using a Lonza Nucleofector 2b and grown for 6 hours post transfection.
|
| Growth protocol |
Neural stem cells were grown in DMEM/F12 with GlutaMAX, StemPro Neural Supplement, bFGF, EGF added in flasks coated with Matrigel at 37°C and 5% CO2. Cells were passaged using Accutase every 48 hours.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Inert and competent libraries were sequenced from plasmid PCR amplicons. Experimental libraries were extracted from NSCs using Qiagen Allprep and MinElute PCR Purification kits (pDNA, 5% of each sample) and Qiagen RNeasy Mini kit (cDNA, 95%). cDNA was synthesized using Invitrogen SuperScript III reverse transcription kit and using a mixture between a custom, library-specific primer and oligo-dT primers (MPRA1) or only custom primer (MPRA2).
We amplfied the library amplicon using qRT-PCR to stop amplification in the exponential phase. pDNA and cDNA libraries were then paired-end sequenced for 150 cycles on an Illumina HiSeq 4000s (MPRA1) or NovaSeq 6000s (MPRA2), including 5% phi-X spike-in.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
MPRA2_cDNA_rep2
Experimental cDNA library barcode tag sequencing
MPRAcounts.tsv.gz
|
| Data processing |
Basecalling performed with CASSAVA
Inert library reads were trimmed using fastx_trimmer (options -Q 33 -f 10) and read pais were aligned using PEAR (option -q 33). Adapter sequences were trimmed using Cutadapt, first the adapters on either side of the construct and finally the construct consisting of enhancer fragment-adapter-barcode tag was split at the adapter into fragment and adapter (option -e 0.16). Fragment sequences were aligned to the fragment library using Bowtie2.
Competent library and experimental MPRA reads containing barcode tags were trimmed using fastx_trimmer (options -Q 33 -l 133 (read 1) -l 126 (read 2) and read pairs were aligned using PEAR (option -q 33). Adapter sequences were trimmed using Cutadapt (option -e 0.16).
ATAC-seq reads were mapped using Bowtie2 (option -X 2000). Reads mapping to the mitochondrion or to ENCODE-defined black listed regions were removed. Peaks were called with MACS2 (options -B --nomodel --shift -25 --extsize 50).
Genome_build: GRCh37/hg19 (ATAC-seq)
Supplementary_files_format_and_content: MPRA count files contain raw counts of MPRA reads mapped to their barcode tag and fragment of origin plus annotations.
|
| |
|
| Submission date |
Nov 25, 2019 |
| Last update date |
Nov 27, 2019 |
| Contact name |
James Patrick Noonan |
| E-mail(s) |
james.noonan@yale.edu
|
| Organization name |
Yale University
|
| Department |
Genetics
|
| Street address |
333 Cedar Street
|
| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06520 |
| Country |
USA |
| |
|
| Platform ID |
GPL27804 |
| Series (1) |
| GSE140983 |
Massively Parallel Reporter Assay of human-specific substitutions in neurodevelopmental enhancers |
|
| Relations |
| BioSample |
SAMN13384350 |
| SRA |
SRX7213995 |
