|
| Status |
Public on Jun 17, 2019 |
| Title |
Pt2A N2 ChIP-seq input rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Chimpanzee neural progenitor cells
|
| Organism |
Pan troglodytes |
| Characteristics |
iPSc line name: Pt2A
cell type: iPSC-derived neural progenitors
developmental stage: N2
|
| Treatment protocol |
For the lentiMPRA, 6 million chimpanzee iPSC-derived neural progenitor cells were plated on a 15 cm dish and cultured for 24 hours. The cells were infected with integrating lentivirus libraries and incubated for 4 days, when they have an estimated 50-100 viral particles/cell. Three independent replicate cultures were infected.
|
| Growth protocol |
http://www.pnas.org/content/107/11/5254.short
Chimpanzee iPSC-derived neural progenitor cells were maintained in N2B27 media supplemented with 20ng/mL bFGF and EGF on matrigel-coated dishes.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cells were washed with PBS three times, and genomic DNA and total RNA was extracted using AllPrep DNA/RNA mini kit (Qiagen). Messenger RNA (mRNA) was purified from the total RNA using Oligotex mRNA mini kit (Qiagen) and treated with Turbo DNase to remove contaminating DNA.
mRNA was reverse transcribed with SuperScript II (Invitrogen) using a primer downstream from the barcode. Resulting cDNA was split into multiple reactions to reduce PCR jack-potting effects and cDNA amplification performed with Kapa Robust polymerase for three cycles, incorporating unique molecular identifiers (UMIs) of 10 bp length. PCR products were cleaned with AMPure XP beads (Beckman Coulter) to remove primers and concentrate samples. These products underwent a second round of amplification in 8 reactions per replicate for 15 cycles, with a reverse primer containing only P7. All reactions were pooled and run on agarose gels for size selection and submitted for sequencing. For DNA, each replicate was amplified for 3 cycles with UMI-incorporating primers, just as the RNA. First round products were cleaned up with AMPure XP beads, and amplified in split reactions, each for 20 cycles. Again, reactions were pooled and gel-purified and sequenced.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
telencephalon
|
| Data processing |
Libraries were sequenced on MiSeq or NextSeq 500 instruments (Illumina) and base called using the instruments' Real Time Analysis softwarre. Multiplexed paired end (PE) Illumina sequencing data was split based on sample barcodes allowing up to one substitution in the barcode sequence.
Paired-end reads shorter or equal to read length were consensus called and adapter trimmed.
To normalize RNA and DNA for different sequencing depths in each sample, we divided reads by the sum of observed counts and reported them as counts per million. Only barcodes observed in RNA and DNA of the same sample were considered.
Barcodes (consensus called paired-end reads) and UMI sequences containing unresolved bases (N) or not matching the designed length of 15bp were excluded. For the HAR control experiments, the control design was combined with the regular HAR design (see human submission). Each barcode x UMI pair was counted only once and only barcodes matching perfectly to those included the oligo design considered. To determine RNA/DNA ratios per insert, we summed up the counts of all barcodes belonging to the same insert and determined the ratio of the average of normalized counts.
ChIP-seq peaks were called using the ENCODE pipeline with default parameters (https://github.com/ENCODE-DCC/chip-seq-pipeline2), aligned to panTro5 and lifted over to GRCh37.
Genome_build: GRCh37
Supplementary_files_format_and_content: All processed data files are in gzip-compressed tab-separated text file format. DNA.tsv.gz and RNA.tsv.gz files contain assigned RNA and DNA barcode counts. HARcontrol_design.tsv.gz contains all additional designed control oligo sequences.
|
| |
|
| Submission date |
Jun 17, 2019 |
| Last update date |
Jun 18, 2019 |
| Contact name |
Jay Shendure |
| Organization name |
University of Washington
|
| Department |
Genome Sciences
|
| Lab |
Shendure
|
| Street address |
3720 15th Ave NE
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195-5065 |
| Country |
USA |
| |
|
| Platform ID |
GPL23423 |
| Series (2) |
| GSE110759 |
Massively parallel dissection of human accelerated regions in human and chimpanzee neural progenitors [chimpanzee] |
| GSE110760 |
Massively parallel dissection of human accelerated regions in human and chimpanzee neural progenitors |
|
| Relations |
| BioSample |
SAMN12078889 |
| SRA |
SRX6078315 |
