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Sample GSM5525318

Query DataSets for GSM5525318

Status

Public on Sep 19, 2021

Title

hiPS_H6_d14_batch1

Sample type

SRA

Source name

hiPS_H6

Organism

Homo sapiens

Characteristics

cell type: human neural forebrain progenitor cells
chip antibody: n/a
days in culture: 14
molecule subtype: polyA RNA

Treatment protocol

Differentiation to fbNPCs: iPSCs were grown to approximately 70-90% confluency and were then dissociated as usual for passaging. After centrifugation, the cells were resuspended in N2 medium (1:1 DMEM/F-12 (21331-020; Gibco) and Neurobasal (21103-049; Gibco) supplemented with 1% N2 (Gibco), 2 mM L-glutamine (Gibco), and 0.2% penicillin/streptomycin). The cells were manually counted twice and plated at a density of 10,000 cells/cm2 in 250 µl medium/cm2 on LN111 Nunc  multidishes (1.14 µg/cm2; Biolamina). 10 µM SB431542 (Axon) and 100 ng/ml noggin (Miltenyi) for dual SMAD inhibition, and 10 µM Y27632 was added to the medium. The medium was changed every 2-3 days (N2 medium with SB431542 and noggin) until day 9 of differentiation, when N2 medium without SMAD inhibitors was used. On day 11, the cells were replated by washing twice with DPBS followed by adding StemPro accutase (75 µl/cm2; Gibco) for 10-20 minutes at 37 ºC. The dissociated cells were washed off with 10 ml wash medium, centrifuged for 5 minutes at 400g and resuspended in B27 medium (Neurobasal supplemented with 1% B27 without vitamin A (Gibco), 2 mM L-glutamine and 0.2% penicillin/streptomycin Y27632 (10 µM), BDNF (20 ng/ml; R&D), and L-ascorbic acid (0.2 mM; Sigma). The cells were counted and replated at 800,000 cells/cm2 on LN111- coated plastic in B27 medium (600 µl medium/cm2). The cells were kept in the same medium until day 14, after which new B27 medium was added (Grassi et al., 2020).

Growth protocol

We used two human iPSC lines generated by mRNA transfection (RBRC-HPS0328 606A1 and RBRC-HPS0360 648A1, both from RIKEN; from here on referred to as HS1 and HS2, respectively). We used two chimpanzee iPSC lines: one generated by mRNA transfection (Sandra A, herein referred to as PT1) and the other with viral vector transduction (PR00818 PTCL-5, herein referred to as PT2) (Marchetto et al., 2013; Mora-Bermúdez et al., 2016). iPSCs were maintained on LN521-coated (0.7 µg/cm2; Biolamina) Nunc multidishes in iPS media (StemMACS iPS-Brew XF and 0.5% penicillin/streptomycin (Gibco)). Cells were passaged 1:2-1:6 every 2-4 days by being rinsed once with DPBS (Gibco) and dissociated using 0.5 mM EDTA (75 µl/cm2; Gibco) at 37 ºC for 7 minutes. Following incubation, EDTA was carefully aspirated from the well and the cells were washed off from the dish using washing medium (9.5 ml DMEM/F-12 (31330-038; Gibco) and 0.5 ml knockout serum replacement (Gibco)). The cells were then centrifuged at 400g for 5 minutes and resuspended in iPS brew medium supplemented with 10 µM Y27632 (Rock inhibitor; Miltenyi) for expansion.

Extracted molecule

polyA RNA

Extraction protocol

On the day of harvest, the cells were washed once with PBS and lysed with 350 µl RLT buffer with 1% mercaptoethanol (Thermo Fisher). RNA was extracted using the RNeasy mini kit (Qiagen) according to manufacturer’s protocol. The quality and concentration of the RNA was analyzed using 2100 Bioanalyzer (RNA nano; Agilent) and Qubit (RNA HS assay kit).
TruSeq RNA Library Prep kit v2 (Illumina)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

iPS-derived human neural forebrain progenitors
RNAseq_H6_C6_map_hg38_pt6.tab
260

Data processing

RNA-seq: raw base call files were converted to fastq and demultiplexed using bcl2fastq. The fastq files were mapped to hg38 and pantro6 reference genomes using STAR/2.5.0a with parameter --outFilterMismatchNoverLmax 0.03 and --sjdbGTFfile gencode.v27.annotation.gtf. When mapping to pantro6, the gencode.v27.annotation.gtf were lifted from hg38 coordinates to pantro6. Gene counts were quantified using featurecounts from Subread/1.5.0-p1, with -s 2, -p and -t exon parameters.
single-cell RNA-seq: raw basecall files were converted to fastq and demultiplexed using cellranger/2.1 mkfastq. Gene counting was done with cellranger count.
Genome_build: hg38
Supplementary_files_format_and_content: counts and coverage tracks

Submission date

Aug 16, 2021

Last update date

Sep 19, 2021

Contact name

Johan Jakobsson

E-mail(s)

johan.jakobsson@med.lu.se

Organization name

Lund University

Department

Wallenberg Neuroscience Center