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Sample GSM4386931

Query DataSets for GSM4386931

Status

Public on Oct 09, 2020

Title

Human1_1_CNCCs

Sample type

SRA

Source name

iPSC-derived CNCCs

Organism

Homo sapiens

Characteristics

cell line: Hu1_1

Growth protocol

Three independent CNCC differentiation experiments were performed. In each one, human-chimpanzee hybrid iPSC (Hy1_30), parental human (Hu1), parental chimpanzee (Ch1) iPSC lines, and human embryonic stem cells (hESCs, control) were differentiated into CNCCs. Briefly, iPSCs and hESCs were incubated with 2mg/ml collagenase for ~30-50 min leading to detachment of colonies. Detached cells were plated as clusters of 100-200 cells in low-attachment petri dishes and cultured in the presence of CNCC differentiation medium consisting of 1:1 Neurobasal medium/DMEM F-12 medium (ThermoFisher Scientific), 0.5× B-27 supplement with Vitamin A (50× stock, GeminiBio), 0.5× N-2 supplement (100× stock, GeminiBio), 20 ng/ml bFGF (Peprotech), 20 ng/ml EGF (Sigma-Aldrich), 5 µg/ml bovine insulin (Sigma-Aldrich) and 1× Glutamax-I supplement (100× stock, ThermoFisher Scientific). Cells grown in CNCC differentiation medium grew as neural spheres/rosettes. For the first four days of differentiation, spheres were separated from cell debris by gentle centrifugation and re-plated into new petri dishes in fresh CNCC differentiation medium. After four days, the neural spheres were allowed to settle for three days to promote attachment to the culture plate surface. After the neural spheres began to attach to the plate, media was changed daily, and neural crest cells were allowed to migrate out of the neural rosettes for 4-5 days. Afterwards, neuroectodermal spheres were manually picked and removed from the culture dishes leaving behind emigrated neural crest cells, which were dissociated with 1x Accutase and passaged onto fibronectin (7.5µg/ml) (ThermoFisher Scientific) coated plates. The early migratory CNCCs were cultured in the presence of maintenance medium comprising of 1:1 Neurobasal medium/DMEM F-12 medium (Invitrogen), 0.5× B-27 supplement with Vitamin A (50× stock, GeminiBio), 0.5× N-2 supplement (100× stock, GeminiBio), 20 ng/ml bFGF (Peprotech), 20 ng/ml EGF (Sigma-Aldrich), 1 mg/ml bovine serum albumin, serum replacement grade (Gemini Bio-Products # 700-104P) and 1× Glutamax-I supplement (100× stock, ThermoFisher Scientific). The CNCCs were cultured on fibronectin coated dishes, with passaging every three days with 1x Accutase for additional two passages. Afterwards, medium was changed to BMP/ChIR medium by adding 3µM ChIRON 99021 (Selleck, CHIR-99021) and 50pg/ml BMP2 (Peprotech) to the maintenance medium, which increased cell proliferation and decreased migration.

Extracted molecule

polyA RNA

Extraction protocol

Qiagen Rneasy Mini Kit with on-column DNAse digestion (Qiagen)
Illumina TruSeq Stranded mRNA library prep kit (Illumina)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Data processing

Remove sequencing adapters with SeqPrep version 1.2
Map reads to GRCh38 and PanTro5 with STAR version 2.5.1b
Remove duplicate reads with Picard 2.18.27
Remove reads which exhibit mapping bias with WASP (Hornet)
Count allele specific and total reads
Genome_build: Human: GRCh38; Chimpanzee: PanTro5
Supplementary_files_format_and_content: Counts matrix with total and allele-specific raw read counts (rows are genes, there are 4 columns per sample: ref counts (aligned to reference genome), alt counts (aligned to alternative genome), no ASE (no segregating SNPs) and ambiguous (SNPs from both genome); matrix text files are provided for samples mapped on both the human and chimpanzee genomes; text files with sample meta data are also included for each data set
Supplementary_files_format_and_content: GRCh38 (1st sheet), panTro5 (2nd sheet)
Supplementary_files_format_and_content: Raw read counts per gene across all samples

Submission date

Mar 05, 2020

Last update date

Oct 09, 2020

Contact name

David Gokhman

E-mail(s)

david.gokhman@mail.huji.ac.il

Organization name

The Hebrew University of Jerusalem

Street address

Givat Ram, Safra Campus