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Sample GSM5577287

Query DataSets for GSM5577287

Status

Public on Feb 04, 2022

Title

H1JA cNESCs P37/D10 6F-2F rep2

Sample type

SRA

Source name

H1 hESCs-derived cNESCs

Organism

Homo sapiens

Characteristics

cell type: cNESCs
growth conditions: Differentiated from hESCs grown in normal growth conditions
treatment: Grown 10 days in 4F medium

Treatment protocol

H1JA and CA1 cNESCs were cultured 10 days in either 6F medium (NES supplemented with 6F) or in 4F medium (NES supplemented with only 10ng/ml FGF2, 3μM CHIR99021, 1μM SB431542, 100nM LDN193189)

Growth protocol

H1 and CA1 human pluripotent stem cells (hPSCs) were routinely cultured on mitomycin C treated mouse embryonic fibroblast feeder cells in KSR media with 10ng/ml FGF2. KSR media contains 80% DMEM-F12 (GIBCO), 20% Knockout serum replacement (GIBCO), 0.1mM 2-mercaptoethanol, 2mM Glutamax, 0.1mM Non-essential amino acids. Prior to neural differentiation hPSCs were transferred to geltrex (GIBCO) coated surface in the absence of feeders and were cultured in mTESR2 (Stem cell technologies) media. On the day of neural induction, hPSCs were replated on geltrex coated surface in N2B27 media supplemented with 10µM SB431542 and 100nM LDN193189 and 10µM Y27632. Neural cultures were detached from Geltrex first on day 8-10 with Accutase or PBS-EDTA, and replated on laminin (Sigma) coated surface, in neural maintenance media (NES) supplemented with 10µM Y27632 at 3x105 cells per cm2 and 6F : 10ng/ml FGF2, 3μM CHIR99021, 1μM SB431542 (Peprotech), 100nM LDN193189, 100nM K02288 (Selleckchem), 100nM AKTiVIII, 75nM MK2206 (Selleckchem), and 1 μM XAV939 (Selleckchem). NES media contains 50% DMEM-F12, 50% Neurobasal, 0.1mM 2mercaptoethanol, 2mM Glutamax, 1x N2 supplement, 0.05x B27 minus vitamin A supplement (GIBCO).

Extracted molecule

polyA RNA

Extraction protocol

Cell lysates were homogenized in QIAshredder columns (QIAGEN #79654) and total RNA was extracted with Rneasy mini columns (QIAGEN). RNA was DNase treated using TURBO DNase (ThermoFisher Scientific #AM2238). RNA quality was verified on a 2100 Bioanalyzer (Agilent Technologies) and polyadenylated RNA was enriched using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB # E7490L)
Libraries were generated using 100 ng of poly(A)+ RNA and Oxford Nanopore Technologies’ Direct cDNA Sequencing Kit (ONT SQK-DCS109), with each sample barcoded with the Native Barcoding Expansion kits (ONT EXP-NBD104 and ONT EXP-NBD114), according to the manufacturer’s specifications. Barcoded samples were pooled, loaded on a MinION sequencer on R9 Flow Cells (ONT FLO-MIN106D), and sequenced using the MinKNOW software v19.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

MinION

Data processing

Raw fast5 files were basecalled using Guppy v.4.2.2 with the guppy_basecaller command and the dna_r9.4.1_450bps_fast.cfg configuration file and default settings. Output fast5 files were generated with the --fast5_out argument.
Barcodes were detected and reads were demultiplexed with the guppy_barcoder using the default settings.
The resulting FASTQ reads were aligned to the GRCh38 human reference with Minimap2 using the following parameters: -aLx splice --cs=long.
Raw read counts were obtained with the featureCounts tool from the Subread package v 2.0.0, using the exon counting mode.
EdgeR R-package (v3.12.1) was then used to normalize the data, calculate RNA abundance (as counts per million reads (CPM), and perform statistical analysis. Briefly, a common biological coefficient of variation (BCV) and dispersion (variance) were estimated based on a negative binomial distribution model. This estimated dispersion value was incorporated into the final EdgeR analysis for differential gene expression, and the generalized linear model (GLM) likelihood ratio test was used for statistics, as described in EdgeR user guide. Differential gene expression was established as genes with a fold change ≥ 1.5, p-value ≤0.05 and FDR ≤0.1.
Genome_build: GRCh38
Supplementary_files_format_and_content: Varga_et_al_lrRNAseq_DE_CPM.txt contains CPM values for each samples obtained from EdgeR, as well as differential expression comparisons for each sets of replicates. EdgeR output contains the log2(Fold Change of expression between samples), log2(CPM), as well as the Likelihood ratio (LR), P-values and False discovery rates (FDR). For more information, please refer to the EdgeR package manual.

Submission date

Sep 14, 2021

Last update date

Feb 04, 2022

Contact name

Samer M.I. Hussein

E-mail(s)

samer.hussein@fmed.ulaval.ca

Organization name

CHU de Québec - Université Laval

Department

Departement of medecine