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| Status |
Public on Nov 30, 2018 |
| Title |
iPSC_AD3 |
| Sample type |
SRA |
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| Source name |
AD3 IPSC
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| Organism |
Homo sapiens |
| Characteristics |
genome build: GRCh38
cell line: AD3-01
condition: IPSC
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| Growth protocol |
Induced pluripotent stem cell generation NHDF1 (from 44-year-old female) was reprogrammed with Yamanaka retroviruses SOX2, KLF4, OCT3/4, c-MYC and NANOG (Takahashi et al., 2006), and has previously been described (Hartfield et al., 2014). AD2-01 (from 51-year-old male) (Buskin et al., in preparation) and AD4-01 (from 68-year-old male) (Melguzo et al, in preparation) were reprogrammed using the CytoTune™-iPS Reprogramming Kit (ThermoFisher). The fibroblasts to generate AD2-01 and AD4-01 were obtained from a commercial source (Lonza, CC-2511). CytoTune-iPS reprogramming was performed as directed by the manufacturer’s instructions (ThermoFisher). The CytoTune reprogramming kit contains four Sendai virus-based reprogramming vectors each capable of expressing one of the four Yamanaka factors (KLF4, OCT3/4, SOX2 and c-MYC). Briefly, after fibroblast transduction with the four Sendai virus-based reprogramming vectors, cells are cultured for 5-6 days, with medium changes every other day (DMEM, high glucose (Sigma), 10% FBS (ThermoFisher), 1% Pen/Strep (100x, ThermoFisher), 200mM L-glutamine (Sigma), 1% non-essential amino acids (ThermoFisher)). The transduced fibroblasts are then passaged using 0.05% Trpsin-EDTA onto pre-prepared feeder layer plates containing mitotically inactivated mouse embryonic fibroblasts (MEF). 3-4 weeks after transduction, colonies should have grown to an appropriate size to allow for manual picking. Using an inverted microscope, a single colony displaying iPSC morphology is cut into 5-6 pieces using a 25 gauge needle, transferred into iPS media (KO-DMEM (ThermoFisher), 25% Knock Serum Replacement (ThermoFisher), 1% nonessential amino acids (100x, ThermoFisher), 200mM L- glutamine (Sigma), 1% Pen/Strep (100x, ThermoFisher), 8 ng/ml human FGF2 (Miltenyi Biotec)) and plated onto pre-prepared MEF plates. Colonies are allowed to attach for 48 hours, and thereafter medium changes are performed daily. iPSCs were adapted to feeder-free conditions onto Matrigel (Scientific Laboratory Supplies)-coated plates in mTeSR1 medium (ScienCell). Bulk passaging was by 0.5 mM ethylenediaminetetraacetic acid (EDTA) to make large-scale, quality-controlled stocks that were cryopreserved in liquid nitrogen. The number of feeder-free passages was kept to a minimum. When selecting iPSCs from frozen stocks for differentiation, vials with the same passage number were selected for each cell line throughout all experiments performed in this study. The iPSC lines AD2-01 and AD4-01 were obtained through the IMI/EU sponsored StemBANCC consortium via the Human Biomaterials Resource Centre, University of Birmingham, UK (http://www.birmingham.ac.uk/facilities/hbrc). All iPSC lines were subject to strict quality control checks before the initiation of differentiation. Quality control checks of this line included: tests for Sendai virus clearance, fluorescence-activated cell sorting (FACS) for pluripotency markers, genomic integrity checks and embryoid body tri-lineage differentiation experiments. Cells are also confirmed as negative for Mycoplasma before cryopreservation. Sensory neuron differentiation For neuronal differentiation, iPSCs were passaged onto Matrigel®-coated six-well plates using TrypLE express (ThermoFisher Scientific) and maintained in mTeSR1 supplemented with 10 μM ROCK inhibitor (ScienCell). Twenty-four hours after plating, the medium was exchanged to mouse embryonic fibroblast (MEF) conditioned medium (ScienCell) supplemented with 10 ng/ml human recombinant FGF2. Cells were allowed to expand on MEF-conditioned medium until ∼50% confluent, at which time differentiation was started according to Chambers et al. (2012). Briefly, medium was exchanged to knockout serum replacement (KSR) medium containing; knockout-DMEM, 15% knockout-serum replacement, 1% GlutaMAX™, 1% non-essential amino acids, 100 μM β-mercaptoethanol, 1% antibiotic/antimycotic (ThermoFisher Scientific), supplemented with the SMAD inhibitors SB431542 (Sigma, 10 μM) and LDN-193189 (Stratech, 100 nM). The medium was gradually transitioned from KSR medium to N2 medium (Neurobasal® medium, 2% B27 supplement, 1% N2 supplement, 1% GlutaMAX™, 1% antibiotic/antimycotic) (ThermoFisher Scientific) over an 11-day period. On Day 2, the small molecules CHIR99021 (Apollo Scientific, 3 μM), SU5402 (R&D Systems, 10 μM) and DAPT (Sigma, 10 μM) were also added. SMAD inhibitors were removed from the media from Day 6 onwards. On Day 11, the now immature neurons were replated onto Matrigel®-coated coverslips (25 000 cells per 13 mm coverslip) in 100% N2 medium containing human recombinant NGF, GDNF, BDNF, NT3 (all at 25 ng/ml, PeproTech) and 10 μM ROCK inhibitor. CHIR99021 (3 μM) was included in the medium until Day 14, and laminin (1 μg/ml, ThermoFisher Scientific) was supplemented into the medium from Day 20 onwards. Medium changes were performed twice weekly after replating. Cytosine β-D-arabinofuranoside (AraC, 2 μM, Sigma) was included in the medium for 24 h following replating to remove the few non-neuronal dividing cells remaining in the culture. This differentiation resulted in a completely pure neuronal culture with extensive arborized neurites by 3 weeks after the end of the small inhibitor stage. Ethics statement Human iPSC lines used in this study were derived from human skin biopsy fibroblasts, following signed informed consent. Three control cells lines were used in this study – AD2-01, AD4-01 and NHDF1. NHDF1 were reprogrammed with approval from research ethics committee: National Health Service, Health Research Authority, NRES Committee South Central, Berkshire, UK (REC 10/H0505/71).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was extracted according to the hybrid method of combined phenol extraction (TriPure, Roche) and column purification (High Pure RNA tissue Kit, Roche) (Bartus et al., 2016). The concentration of RNA in the samples was measured using a nanodrop and proved sufficient for sequencing. According to the hybrid method, DRG tissue was first homogenized in TriPure and then mixed with chloroform, following the phenol extraction method. After centrifuging the aqueous liquid, which stays on the top of the tube and contains the nucleic acids, was removed. This solution was then subjected to the column purification method. Following this protocol, the clear aqueous liquid was placed in Roche High Pure RNA tissue Kit columns and washed several times in order to purify the RNA. RNA was then extracted with the mRNeasy kit and all samples were subjected to on-column DNAse digestion in order to prevent genomic contamination i.e. presence of DNA in the RNA samples. RNA libraries were prepared for sequencing using standard Illumina protocols. Second strand cDNA synthesis incorporated dUTP. The cDNA was end-repaired, A-tailed and adapter-ligated. Prior to amplification, samples underwent uridine digestion. The prepared libraries were size selected, multiplexed and QC’ed before paired end sequencing over eight lanes of a flow cell.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Poly-A selected RNA
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| Data processing |
Sequencing, base calling, demultiplexing and QC using Illumina HiSeq4000 analysis software version HD 3.4.0.38 Demux of files and FastQ preparation by BcltoFastq 2.17.1.14 Reads were mapped to GRCh38 human genome (Homo_sapiens.GRCh38.dna.primary_assembly.fa, Homo_sapiens.GRCh38.88.chr.gtf) using STAR aligner BAM files were merged across sequencing lanes and sorted using SAMtools Features were counted using HTSeq with the intersection not empty strategy All reads overlapping annotations: RefSeq (Pruitt et al., 2014), XenoRefSeq, ENSEMBL genes (Harrow et al., 2012) were discarded. We then used the remaining subset of RNA-seq reads in order to identify islands of expression outside known gene models (continuously expressed regions) using a coverage window approach. Workflow generally described in (Cabili et al., 2011; Gerstein et al., 2014; Ilott and Ponting, 2013) with modifications to produce annotations at the gene level. Doing this we get a non-redundant annotation of unique genes of LncRNAs suitable for count based DE analysis (Anders et al., 2015; Love et al., 2014). The concept of islands of expression (I.o.E) is described in (Gerstein et al., 2014). Coverage cut-off has been set as in (Cabili et al., 2011) Only properly paired and uniquely mapped reads were selected. We selected splicing junctions covered with > 2 reads. We discarded all reads overlapping know gene models. We then used the remaining subset of RNA-seq reads to identify I.o.E outside known gene models using a coverage window approach. We selected continuous regions above the coverage threshold of more than a read-mate length to ensure that overlapping read-mates would not artificially increase coverage. I.o.E were collapsed and clustered as co-overlapping features connected by splicing junctions. In each cluster consensus introns were calculated by the relative frequency of each discrete segment of a set of splicing junctions. We then subtracted the genomic intervals of these consensus introns from the genomic intervals of the grouped (I.o.E) to reconstruct full length putative LncRNAs. We included putative LncRNAs in this novel annotation only if they were present in all replicates a biological condition or strain. Annotations were exported in the Gene Transfer Format (GTF). Subsequently we filtered out transcripts with length < 200 bp and we used CPAT (Wang et al., 2013) to assess coding potential. The pipeline was scripted in R using bioconductor (Gentleman et al., 2004) packages and custom scripts.
Genome_build: GRCh38
Supplementary_files_format_and_content: CSV files *res*.csv include DESeq2 results for both ENSEMBL genes (ens_genes_res) and novel LncRNAs (lncs_res). CSV files *toc*.csv include read-pairs counts calculated using HTSeq. GTF files Novel_LncRNAs_*.gtf included gene models of novel LncRNAs.
Supplementary_files_format_and_content: TOC.csv: abundance measurements in IPSC and IPS derived sensory neurons
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| Submission date |
Nov 29, 2018 |
| Last update date |
Nov 30, 2018 |
| Contact name |
Georgios Baskozos |
| E-mail(s) |
georgios.baskozos@ndcn.ox.ac.uk
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| Phone |
07428256551
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| Organization name |
Oxford University
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| Department |
NDCN
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| Lab |
Neural Injury Group
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| Street address |
Level 6, West Wing, John Radcliffe Hospital
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| City |
Oxford |
| ZIP/Postal code |
OX3 9DU |
| Country |
United Kingdom |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE107181 |
Comprehensive analysis of Long non-coding RNA expression in dorsal root ganglion reveals cell type specificity and dysregulation following nerve injury [human iPS] |
| GSE107182 |
Comprehensive analysis of Long non-coding RNA expression in dorsal root ganglion reveals cell type specificity and dysregulation following nerve injury |
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| Relations |
| BioSample |
SAMN10498873 |
| SRA |
SRX5079889 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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