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Sample GSM4743057

Query DataSets for GSM4743057

Status

Public on Jan 06, 2021

Title

NPC mRNA-seq

Sample type

SRA

Source name

induced pluripotent stem cell

Organism

Homo sapiens

Characteristics

cell type: Human induced pluripotent stem cell derived
developmental stage: Neuronal progenitor cell

Treatment protocol

For neuronal differentiation, an embryoid body (EB) was generated by culturing human iPSCs for 5-6 days on non-adherent Petri dishes in hiPSC maintain medium absence of bFGF. To guide differentiation of EBs into neuroectodermal fate, additional supplement of 5μM dorsomorphin (DM) (Sigma-Aldrich) and 5μM SB431542 (Sigma-Aldrich) were added to the hiPSC medium during EB formation. EBs were then attached to the culture dish coated with Matrigel (BD Biosciences) and were cultured in neural induction medium consist of DMEM/F12 medium (Invitrogen), 1×N2 supplement (Invitrogen), 1×nonessential amino acids (Invitrogen) for 6 days10-12. When neural rosettes appear in the center of the EBs, then NPCs were collected through manipulated Pasteur pipet. Collected NPCs were re-plated on Matrigel coated plate for neuronal maturation and cultured for 14 days in Neurobasal medium (Invitrogen) containing 1X B27 supplement without vitamin A (Invitrogen), 1x Glutamax (Invitrogen) supplemented with 10 ng/ml brain-derived neurotrophic factor (BDNF, Peprotech), 10 ng/ml glial cell-derived neurotrophic factor (GDNF, Peprotech), 10 ng/ml neurotrophin-3 (NT3, Peprotech). Early neurons were collected by Accutase (Invitrogen) treatment.

Growth protocol

Human iPS Cell line were kindly provided from Yonsei University College of Medicine. First, STO fibroblast (American Type Culture Collection (ATCC)) was cultured and inactivated by mitomycin C (10μg/ml, Sigma-Aldrich) for feeder cell preparation. iPSCs were cultured on feeder cell layers and maintained using hiPSC maintain medium consist of DMEM/F12 (Invitrogen, Carlsbad, CA, USA), 20% knockoutserum replacement (Invitrogen), 1% penicillin-streptomycin (Invitrogen), 1×non-essential amino acid (Invitrogen), 0.1mM β-mercaptoethanol (Sigma), and 10ng/ml basic fibroblast growth factor (bFGF) (Peprotech).

Extracted molecule

total RNA

Extraction protocol

Genomic DNA was extracted by PureLink™ Genomic DNA Kit according to manufacturer’s protocol. Total RNA of each cell type was extracted by TRIzol™ Reagent (Invitrogen) and RNA integrity (RIN) was assessed by Agilent Bioanalyzer (RIN>=8). 1 million cells were crosslinked in crosslinking buffer (100 mM NaCl, 0.1mM EDTA, 5mM Hepes, pH 8.0, 1% formaldehyde) for 10 min at 25°C. The crosslinking was quenched with 125 mM glycine in 25°C for 5 min with rotation and washed twice with ice-cold PBS. The cross-linked cells were re-suspended in SDS lysis buffer (1% SDS, 50 mM Tris-HCl, pH8.0, 10mM EDTA) with protease inhibitor (Roche). Mono- and di-nucleosome size chromatin was obtained through sonication (Covaris, S220)
Total 300ng of genomic DNA was used for bisulfite conversion (EZ DNA Methylation-Gold Kit, Zymo research) and library preparation (TruSeq DNA Methylation, Illumina). We sequenced the bisulfite converted genomic library (2x150bp) using HiseqX. 200ng of total RNA was used for library preparation with TruSeq rapid SBS kit or Truseq SBS Kit v4 according to the kit guidelines. We sequenced the paired-end RNA-sequencing library (2x101bp) using HiSeq 2500 system. The immunoprecipitated DNA was recovered using AMPure XP beads (Beckman Coulter) and ChIP-seq libraries were prepared using NEBNext Ultra II DNA library Prep Kit following the manufacturer’s instruction. The ChIP-seq libraries were sequenced in 150 bp paired-end mode using Illumina NovaSeq 6000 platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

iPSC_NPC_starrsem_hg19.txt

Data processing

We used trim-galore (v.0.4.0) to trim off the adapter sequences or low-quality reads. Trimmed sequences were then aligned to the human reference genome (hg19) using Bismark (0.14.4) with –bowtie2 option. Duplicated reads were removed using Picard markduplicate (1.141). Methylated value (Beta value, the ratio of intensities between methylated and un-methylated alleles) for all the covered CpG sites were calculated using bismark_methylation_extractor (0.14.4) “–no_overlap –comprehensive –bedgraph”.
Raw reads were aligned to the human reference (hg19) using STAR (v2.6.0a) with the support of transcriptome reference (GRCh37, UCSC). From the mapped reads, we further calculated the TPM (Transcripts per Million) using RSEM (v1.2.31) at the gene level. To reduce any technical bias among the samples, we further quantile normalized using R library, preprocesscore.
For read alignment, we used BWA (0.7.7) to map reads to the human genome sequence (hg19). We further removed duplicated reads using Picard MarkDuplicate (1.141). For peak calling, we used MACS2 (2.1.0) to call the significantly enriched ChIP region compare to the input IgG control “q -0.01 (FDR <0.01%)”
Genome_build: hg19
Supplementary_files_format_and_content: abundance measurements
Supplementary_files_format_and_content: peak calling data

Submission date

Aug 24, 2020

Last update date

Jan 06, 2021

Contact name

Young-Joon Kim

E-mail(s)

yjkim@yonsei.ac.kr

Organization name

Yonsei university

Street address

Yonsei-ro 50