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Sample GSM4135906 Query DataSets for GSM4135906
Status Public on Jan 21, 2020
Title iSN_Bioreplicate_3
Sample type SRA
 
Source name sensory neurons induced by NGN2-BRN3A expression in iPSCs (iSN)
Organism Homo sapiens
Characteristics biological replicate: 3
sample group: iSN
donor: X
age: 30
gender: Male
race: Japanese
Treatment protocol NA
Growth protocol The hDRG was purchased as a pooled RNA sample (Clontech, 636150). The iPSC, iSN, NC-iSN1, and NC-iSN2 samples were all derived from the WTC11 control iPSCs, an iPSC line generated previously (Miyaoka et al., 2014). iPSCs were maintained on tissue culture-treated polystyrene plates with Matrigel (Corning, 354277) in E8 medium (Thermo Fisher, A1517001). The medium was changed daily and the iPSCs were split every 4 – 6 days using Accutase (Invitrogen, A1110501). The rho kinase (ROCK) inhibitor 10 µM Y-27632 (Tocris, 1254) was included in the medium on the day of passaging. To generate iSN samples, on day 0 iPSCs were dissociated with Accutase, and 20,000 cells/cm2 were plated on Matrigel-coated dishes in neural differentiation medium (NDM) supplemented with 10 µM Y-27632 and 2µg/mL doxycycline (Clontech, 631311). NDM (all from Invitrogen) consisted of equal parts DMEM/F12 (11330032) and Neurobasal medium (21103049), containing GlutaMAX (35050061), B27 (17504044), and N2 (17502048). The medium could be optionally changed on day 1 to remove Y-27632, but is otherwise the same composition. On day 2, the cells were dissociated with Accutase, and 50,000 cells/cm2 were seeded on plates coated with 0.1% polyethylenimine (Sigma, P3143) and 15 µg/mL laminin (Invitrogen, 23017015) in NDM with doxycycline. The medium was changed every other day through day 8, after which only half volume medium changes were made. If undifferentiated cells persist, 20 µM BrdU could be optionally added on day 4 to eliminate dividing cells and achieve a pure neuronal culture. Starting on day 8 onward, neurotrophic factors (NTFs) (all R&D Systems) were added to the NDM, consisting of BDNF (248BD025), GDNF (212GD010), β-NGF (256GF100), and NT-3 (267N3025), each at 10 ng/mL. Doxycycline was discontinued starting on day 14. Cells were collected for analysis on day 21. To generate NC-iSN1 and NC-iSN2 samples, the neural crest differentiation was a modified form of previous protocols (Bajpai et al., 2010; Schrenk-Siemens et al., 2015). On day -1 iPSCs were dissociated with Accutase and seeded in an Aggrewell 400 plate for spheroid formation (STEMCELL Technologies, 34411) according to manufacturer’s instructions in E8 medium with 10 µM Y-27632. On day 0, spheroids were transferred to uncoated tissue culture-treated dishes in NDM with 20 µg/mL bFGF (R&D Systems, 233FB025), 20 µg/mL EGF (R&D Systems, 236EG200), and 10 µM of the TGF-β receptor ALK4/5/7 inhibitor SB431542 (Tocris, 1614). This medium was changed every other day. SB431542 was discontinued starting on day 6. After 1-2 weeks, spheroids spontaneously attached to the culture surface and neural crest cells migrated outward. All spheroids were manually removed, and the neural crest cells were re-plated at 40,000 cells/cm2 on polyethylenimine and laminin-coated dishes in NDM – this is denoted as day 0 of neural induction. For NC-iSN1s, doxycycline was added from day 0 until day 14, and NTFs were supplemented beginning on day 8. For NC-iSN2s, doxycycline was added on day 0 for 24 hours. NTFs and 50 nM retinoic acid (Sigma, R2625) were supplemented from day 0 onwards. On day 2, the neural crest cells were re-plated for the final time onto polyethylenimine and laminin-coated dishes at 50,000 cells/cm2. Medium was changed every other day, and half medium changes were made after day 8. Cells were collected for analysis on day 21.
Extracted molecule total RNA
Extraction protocol RNA was extracted from iPSCs and iSNs using TRIzol (Thermo Fisher, 15596018) and chloroform and purified with an miRNeasy kit (Qiagen, 217004) following manufacturer’s instructions. Total RNA quantity and integrity were assessed on a Bioanalyzer (Agilent).
For the iSN samples, 500 ng total RNA was used in conjunction with the TruSeq Stranded Total RNA Library Prep kit (Illumina) to prepare libraries. The library quality was checked via Bioanalyzer and quantitated by Qubit (Thermo Fisher). Equimolar quantities from each library were pooled and run on a NextSeq 500/550 High Output kit v2.5 (150 cycles) (Illumina, 20024907) at approximately 40 million paired-end reads per sample. For the hDRG (Clontech 636150) and iPSCs, sequencing libraries were constructed from 100 ng – 500 ng of total RNA using the TruSeq Stranded Total RNA kit (Illumina) with Ribo-Zero following the manufacturer’s instructions. The fragment size of the libraries was verified using the Agilent 2100 Bioanalyzer (Agilent) and the concentrations were determined using Qubit instrument (Thermo Fisher). The libraries were loaded onto the Illumina HiSeq 3000 for 2 x 75 base pair paired-end read sequencing at approximately 40 million reads per sample.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 3000
 
Description sensory neurons induced by NGN2-BRN3A expression in iPSCs (iSN)
Data processing RTA 1.13.48 and CASAVA 1.8.2. software used for basecalling and deplexing.
Raw sequence files generated per library were quality inspected using the FastQC tool (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) then adaptor clipped (TruSeq3-PE-2.fa:2:30:10) and trimmed to remove 5' nucleotide bias (HEADCROP:15) and low quality calls (TRAILING:20 SLIDINGWINDOW:4:20 MINLEN:15) using the Trimmomatic tool (http://www.usadellab.org/cms/?page=trimmomatic). Surviving intact pairs of reads per library were then reference mapped against the current instance of the human genome (GRCh38.82) using the RNA-Seq tool supported in v11 of the CLCbio Genomics Workbench (www.qiagenbioinformatics.com). After, expression per known annotated gene (Homo_sapiens.GRCh38.82.chr.gtf) were enumerated and exported in Transcripts Per Kilobase Million (TPM) units.
Genome_build: GRCh38.82
Supplementary_files_format_and_content: One tab-delimited text file per sequenced library are provided describing the observed gene expression in Transcript Per Kilobase Million (TPM) units.
 
Submission date Oct 23, 2019
Last update date Jan 22, 2020
Contact name Kory R Johnson
E-mail(s) johnsonko@ninds.nih.gov
Phone 301-402-1956
Organization name NINDS/NIH
Department DIR IT & Bioinformatics
Lab Bioinformatics Section
Street address 10/3B01, 9000 Rockville Pike
City Bethesda
State/province MD
ZIP/Postal code 20892
Country USA
 
Platform ID GPL21290
Series (1)
GSE139273 Transcriptional Programming of Human Mechanosensory Neuron Subtypes from Pluripotent Stem Cells
Relations
BioSample SAMN13091387
SRA SRX7043715

Supplementary file Size Download File type/resource
GSM4135906_iSN_Bioreplicate_3_hg38_GeneLevel_TPM_Expression.txt.gz 2.5 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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