GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2211639

Query DataSets for GSM2211639

Status

Public on Aug 07, 2016

Title

Chimp PR00818 NPC A

Sample type

SRA

Source name

NPCs derived from PR00818 iPSC line

Organism

Pan troglodytes

Characteristics

cell type: neural progenitor cells (NPCs)
origin: PR00818 induced pluripotent stem cell (iPSC) line

Growth protocol

Human iPSC lines (WT-33 and ADRC-40) and chimpanzee iPSC lines (PR00818 and PR01209) have been previously described (Marchetto et al. Nature 2013, GEO GSE47626). To obtain NPCs from human and chimpanzee iPSCs, embryoid bodies (EBs) were formed by mechanical dissociation of iPSC clusters and plated into low-adherence dishes in DMEM/F12 plus N2 and B27 (Invitrogen) medium in the presence of Noggin (R&D) for forebrain induction for approximately 7 days. Then, floating EBs were plated onto poly-ornithine/laminin (Sigma)-coated dishes in DMEM/F12 plus N2 and B27 (Invitrogen) with addition of Noggin. Rosettes were visible to collect after 7 days. Rosettes were then dissociated with accutase (Chemicon) and plated again onto coated dishes in DMEM/F12 plus N2 and B27 and 10ng/ml of FGF2 (R&D). Homogeneous populations of NPCs were achieved after 1-2 passages with accutase in the same conditions. To obtain mature neurons, NPCs were cultured with DMEM/F12 plus N2 and B27 with addition of 1ug/ml of Laminin, BDNF (20 ng/ml), GDNF (20 ng/ml) and cyclic AMP (500 ug/ml) for 8 weeks.

Extracted molecule

total RNA

Extraction protocol

Total cellular RNA was extracted from ~1-5x10^6 cells using the RNeasy Protect Mini kit or RNeasy Plus kit (Qiagen, Valencia, CA), according to the manufacturer's instructions.
For RNA library generation from human and chimpanzee NPCs and eight-week-old neurons, polyA+ RNA was fragmented and prepared into sequencing libraries using the Illumina TruSeq RNA sample preparation kit. NPC-derived sequencing libraries were analyzed on an Illumina HiSeq 2000 sequencer at the UCSD Biomedical Genomics Laboratory (BIOGEM). cDNA libraries were prepared from two human and two chimpanzee NPC lines (two clones each) derived from human WT-33 and ADRC-40 iPSC lines and chimpanzee PR00818 and PR01209 iPSC lines, respectively. Libraries were sequenced using single-end 100 bp reads at a depth of 15-30 million reads per library. Sequencing libraries derived from eight-week-old neurons were analyzed on an Illumina HiSeq 2500 sequencer at the Salk Next Generation Sequencing Core. cDNA libraries were prepared from two human (WT-33 and ADRC-40) and two chimpanzee (PR00818 and PR01209) neurons, one clone each. Libraries were sequenced using paired-end 100 bp reads at a depth of 15-30 million reads per library.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

PolyA+ RNA-Seq (not strand-specific)
Processed data file: TPM_NPC_Neuron_hg19.txt

Data processing

Reads from all libraries were mapped to both the human (hg19, GRCh37) and chimpanzee (panTro4, CGSC 2.1.3) genomes using STAR (v2.2.0c). Due to the lack of annotation in the chimpanzee genome, human gene models (RefSeq) were used to quantify gene expression. To avoid bias introduced by genome insertions and deletions, only reads mapping to both the human and chimpanzee genomes uniquely were used from each sample when comparing gene expression values (~4% of reads mapped to only one genome per sample). To calculate gene expression, read counts in the exons of RefSeq transcripts were calculated using HOMER.
Gene expression was calculated in TPM with Kallisto (version 0.42.1) against a custom catalog of human transcripts, including all human RefSeq transcripts with the three BOLA2 isoforms. RefSeq isoforms nearly identical to the canonical BOLA2 isoform (NM_001039182 and NM_001031827) were not included in the catalog of transcripts.
Since the RNA-seq datasets are a mix of PE100 and SE100 reads, we quantified gene expression by using only the first read of PE100 sequencing.
Genome_build: hg19 (GRCh37)
Genome_build: panTro4 (Pan_troglodytes-2.1.4)
Supplementary_files_format_and_content: TPM_NPC_Neuron_hg19.txt: Tab-delimited text file with TPM values from all experiments, RefSeq based with custom bola2 transcripts, and gene annotation information for RefSeq genes BOLA2 characterized isoforms (hg19 assembly).

Submission date

Jun 22, 2016

Last update date

May 15, 2019

Contact name

Inigo Narvaiza

E-mail(s)

narvaiza@salk.edu

Organization name

Salk Institute

Department

Laboratory of Genetics