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| Status |
Public on Jun 25, 2024 |
| Title |
cNSC_RNAseq_rep3 |
| Sample type |
SRA |
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| Source name |
C8861
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| Organism |
Pan troglodytes |
| Characteristics |
cell line: C8861
cell type: Neural Stem Cells derived from IPSCs
cut &_run_antibody: NA
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| Growth protocol |
Detailed information on growth and differentiation protocols is provided in the manuscript
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction done using Qiagen RNeasy Plus Mini Kit (Cat # 74134)
Libraries were prepared and sequenced (2×150 bp) using standard Illumina protocols on an Illumina NovaSeq 6000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
KAPA mRNA HyperPrep kit for standard input PolyA library prep
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| Data processing |
CHi-C: Ligated reads were truncated, mapped and filtered to DpnII digested reference genomes (generated from the GRCh38 and panTro6 reference assemblies for human and chimpanzee respectively) using the HiCUP pipeline (truncating RE site flag = ^GATC,DpnII; mapping flags for Bowtie2: --very-sensitive, --no_unal, --reorder; ditag pairing criteria: MAPQ score >= 30, XS – AS tag ID score >= 10; filtering ditag range = 50 to 800bp). Valid ditags were deduplicated using the adapter UMIs through the umitools package. All DpnII fragments containing HARs and HGEs were then merged into a single bait file and significant interactions were called using CHiCAGO after combining 3 replicates from the HiCUP pipeline. Briefly, we weighted the 4-parameter Delaporte model in CHiCAGO using parameters: weight(alpha) = 24.5, weight(beta) = -2.16, weight(gamma) = -21.2 and weight(delta) = -9.2, and this was used to compute weighted P-values for every interaction. We then set a threshold for the CHiCAGO scores as >4.5 for human and >4.4 for chimpanzee samples. CUT&RUN: Reads were trimmed for adapters using Trimmomatic and aligned to either the GRCh38 (human) or panTro6 (chimpanzee) genomes using Bowtie2 (flags: --very-sensitive, -X 2000). Aligned reads were filtered based on quality and PCR duplicates were marked using the MarkDuplicates function in Picard (courtesy the Broad Institute bioinformatic toolkit). Reads with the following flags – “reads unmapped”, “not primary alignment reads” and “failing platform duplicates” were removed and the remaining properly mapped reads were used for peak calling using the program SEACR (with the --stringent flag), which was used to call enriched regions in target data by selecting the top 0.5-1% of regions by AUC depending on the mark assayed. RNA-Seq: For the hNSC versus cNSC comparison, paired-end reads were mapped to the XSAnno whole transcriptome annotation modified to compare GRCh38 to panTro6 genomes on the GENCODEv43 annotation. Briefly, this annotation subsets out genes with exons that reciprocally liftover between both species (>98% sequence similarity), then filters out those with ambiguous annotations using BLAT [blat parameters: % ID between species = 95%, % len between species = 95%, % ID within species (paralogs) = 97%, % len within species = 97%] and finally simulates reads and filters exons that are differentially expressed in the simulation using DESeq2. Mapping was performed using the STAR aligner (flags: --outSAMunMapped Within, --twopassMode Basic, --outFilterMultimapNmax 1, --quantMode TranscriptomeSAM). Using the package RSEM we generated a counts matrix for each species’ replicate and then used DESeq2 for differential gene expression analysis. Genes with FDR-adjusted P-value < 0.01 and |log2FC| > 1 were considered differentially expressed between human and chimpanzee. For the hNSC- human neuron comparison, reads were mapped to native GRCh38 RefSeq coordinates and TPM counts from RSEM in each cell type were compared for specific gene targets. scRNA-Seq: Reads were aligned to GRCh38 using the multi function of CellRanger 6.0.1. For quality control, cells with nFeature_RNA (number of genes) and/or nCount_RNA (number of molecules) values 2 standard deviations away from the mean were removed, alongside cells with mitochondrial reads greater than 10% of total reads. Counts were normalized using Seurat v4.3.0, via the NormalizeData function with default parameters. Human and Chimpanzee count matrices were then integrated using the SelectIntegrationFeautres, FindIntegrationAnchors, and IntegrateData functions with default parameters. Dimensionality reduction was performed using RunPCA(params: npcs = 30) and RunUMAP on the integrated data, and Louvain clustering was performed using FindNeighbors(params: dims = 1:15) and FindClusters(params: resolution = 0.3). Cluster identities were assigned by identifying cell-type markers using FindAllMarkers(params: min.pct = 025).
Assembly: Human: GRCh38, chimpanzee: panTro6
Supplementary files format and content: C-HiC: Output txt files from HiCUP pipeline based on aligned and deduplicated Capture HiC reads linked to HARs and HGEs (one per replicate), which can be input for CHiCAGO to call significant interactions; CUT& RUN: bigwig and bed files with signal and peak calls for each chromatin mark
Supplementary files format and content: RNA-Seq: Output txt files post transcript quantification by RSEM containing raw counts, FPKM and TPM values per replicate. hNSC and cNSC processed files are in XSAnno human-chimpanzee consensus coordinates based on GENCODEv43, human neuron processed files are in native GRCh38 RefSeq coordinates; scRNA-Seq: txt files with feature, barcodes, matrix information for each species separately (demultiplexed)
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| Submission date |
Jun 20, 2024 |
| Last update date |
Jun 25, 2024 |
| Contact name |
Atreyo Pal |
| E-mail(s) |
atreyo.pal@yale.edu
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| Organization name |
Yale University
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| Department |
Genetics
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| Lab |
Noonan
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| Street address |
333 Cedar St
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| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06510 |
| Country |
USA |
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| Platform ID |
GPL30573 |
| Series (1) |
| GSE270272 |
Resolving the three-dimensional interactome of Human Accelerated Regions during human and chimpanzee neurodevelopment |
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| Relations |
| BioSample |
SAMN41922424 |
| SRA |
SRX24983683 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8338677_cNSC_RNAseq_rep3.genes.results.gz |
1.6 Mb |
(ftp)(http) |
RESULTS |
SRA Run Selector |
| Raw data are available in SRA |
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