{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Zane Kliesmete"],"organism":["Macaca fascicularis"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15695"],"description":["Single cell RNA-seq samples from 3 species across a differentiation from induced pluripotent stem cells to neural progenitor cells were generated to study gene expression evolution.  Briefly, previously generated urinary stem cell derived iPSCs of 3 human (Homo sapiens) individuals (3 clones), 1 gorilla (Gorilla gorilla) individual (2 clones) and fibroblast derived cynomolgus macaque (Macaca fascicularis) iPSCs of 2 individuals (4 clones) (Geuder et al. 2021) were differentiated to neural progenitor cells via dual-SMAD inhibition as three-dimensional aggregation culture (Chambers et al. 2009; Ohnuki et al. 2014). Single cell RNA-seq libraries from differentiation days 0, 1, 3, 5, 7, 9 were generated using mcSCRB-seq protocol (Bagnoli et al. 2018)."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - Libraries were prepared using mcSCRB-seq (Bagnoli et al., 2018). The method was followed exactly as outlined in the step-by-step protocol (Bagnoli et al., 2018) with the exception of using 17 cycles for the pre-amplification.","Sample Collection - Primate iPSCs were cultured in StemFit + bFGF as described previously (Geuder et al., 2021). For differentiation, the cells were dissociated and 9 × 103 cells were plated into each well of a low attachment U-bottom 96-well-plate in 8GMK medium consisting of GMEM (Thermo Fisher), 8% KSR (Thermo Fisher), 5.5 ml 100 × NEAA (Thermo Fisher), 100 mM Sodium Pyruvate (Thermo Fisher), 50 mM 2-Mercaptoethanol (Thermo Fisher) supplemented with 500 nM A-83–01 (Sigma Aldrich), 100 nM LDN 193189 (Sigma Aldrich) and 30 µM Y27632 (biozol). Medium was changed every second day.  Spheres were collected in a reaction tube, washed with PBS and fixed with 4% PFA. After the spheres were incubated in 10%, 20% and 30% sucrose solution, they were stored at -80°C until further processing. Embedded spheres were then cut using a cryostat to slices a 20 µm. For staining the slides were thawed, rinsed with PBSand incubated with 0.5% TritionX100 for one hour. Primary antibodies in blocking solution were subseqently incubated overnight at 4°C. After washing with 0.05 % TritonX100/PBS at 37°C for 30 minutes, secondary antibody and DAPI were added and incubated overnight. After another 0.05 % TritonX100/PBS treatment at 37°C for 30 minutes, slides were mounted using vectashield and imaged on a confocal microscope.","Sequencing - Libraries were paired-end sequenced on an Illumina HiSeq 1500 instrument. Sixteen bases were sequenced with the first read to obtain cellular and molecular barcodes and 100 bases were sequenced in the second read into the cDNA fragment,","Nucleic Acid Extraction - On day 0,1,3,5,7 and 9 cells were dissociated using accumax and single cell sorted using a BD FACS Aria II. The cells were sorted into 96-well plates containing lysis-buffer consisting of Phusion buffer, proteinase K and barcoded oligo-dT primers."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - FASTQ data was processed using the zUMIs pipeline (Parekh, Ziegenhain, et al., 2018) that utilizes STAR aligner (Dobin et al. 2013). We mapped all reads to 3 reference genomes: Homo sapiens Hg38 (GENCODE release 32), Gorilla gorilla Kamilah GGO v0/gorGor6 (UCSC), and Macaca fascicularis macFas6 (ENSEMBL release 109). To reduce computational time, we removed contigs smaller than 150 kb from the gorGor6 genome. The gorilla and macaque GTF files were created by liftoff of the hg38 annotation to the corresponding primate genomes (Shumate et al., 2021), followed by removal of transcripts with partial mapping (<50%), low sequence identity (<50%) or excessive length (>100 bp difference and >2 length ratio). We run zUMIs using the STAR parameters –limitOutSJcollapsed 2000000 –limitIObufferSize 300000000 –limitSjdbInsertNsj 10000000, a pre-given cell barcode list, exon-only counting (introns = no) and positive strandedness (strand = 1)"],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina HiSeq 1500"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Macaca fascicularis"],"pubmed_authors":["Zane Kliesmete"],"additional_accession":[]},"is_claimable":false,"name":"Single cell RNA-seq of of human (Homo sapiens), gorilla (Gorilla gorilla) and cynomolgus macaque (Macaca fascicularis) iPSC and iPSC-derived NPC cell lines","description":"Single cell RNA-seq samples from 3 species across a differentiation from induced pluripotent stem cells to neural progenitor cells were generated to study gene expression evolution.  Briefly, previously generated urinary stem cell derived iPSCs of 3 human (Homo sapiens) individuals (3 clones), 1 gorilla (Gorilla gorilla) individual (2 clones) and fibroblast derived cynomolgus macaque (Macaca fascicularis) iPSCs of 2 individuals (4 clones) (Geuder et al. 2021) were differentiated to neural progenitor cells via dual-SMAD inhibition as three-dimensional aggregation culture (Chambers et al. 2009; Ohnuki et al. 2014). Single cell RNA-seq libraries from differentiation days 0, 1, 3, 5, 7, 9 were generated using mcSCRB-seq protocol (Bagnoli et al. 2018).","dates":{"release":"2025-12-10T00:00:00Z","modification":"2026-05-27T16:12:23.56Z","creation":"2025-12-12T23:13:37.189Z"},"accession":"E-MTAB-15695","cross_references":{"ENA":["ERP185490"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005684","EFO_0005518","EFO_0003816","EFO_0004184"]}}