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| Status |
Public on Sep 17, 2021 |
| Title |
RRBS_iNSC 3, rep 1, Gibco protocol |
| Sample type |
SRA |
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| Source name |
Dura mater
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| Organism |
Mus musculus |
| Characteristics |
strain: C57Black6/J
tissue: Dura mater
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| Treatment protocol |
Fibroblasts cultures were established from small strips of dura mater, collected at surgery. Tissue was minced with a scalpel then spun down and resuspended in trypsin for 5 minutes at 37 degrees. To stop the reaction fresh fibroblast media (DMEM, Glutamax, 10% Foetal calf serum, 2% L-Glutamin and 1% penicillin-streptomycin) was added. Samples were centrifuged and resuspended in fresh media, then plated in 6 well plates (Corning #BC010). Media was topped up frequently during the first week then changed every other day. Fibroblasts reprogramming was performed following a previously described protocol. Briefly, fibroblasts were electroporated with reprogramming vectors expressing Oct4, c-Myc, Klf4, Sox2 (OCKS 4F, 5µg) and Rarg, Lfh1 (RL 2F, 5.0 µg) using Amaxa Nucleofector (Lonza, Germany) then plated on SNL feeders with M15 media (Knockout DMEM Invitrogen, 15% Fetal Bovine Serum Hyclone, 1X Glutatamin-Penicillin-Streptomycin Invitrogen, 1X non-essential amino acids Invitrogen). Upon the appearance of colonies, media was replaced with EPSCM (DMEM/F12 Invitrogen, 20% Knockout Serum Replacement Invitrogen, 1X Glutamin-Penicillin-Streptomycin, 1X non-essential amino acids Invitrogen, 0.1 mM β Mercapto-ethanol Sigma, 106 U/ml hLIF Millipore supplemented with the following inhibitors: CHI99021 Tocris 1µM, JNK Inhibitor VIII Tocris 4 µM, SB203580 Tocris 10µM, A-419259 Santa Cruz 1µM and XAV939 Stratech 1µM). Colonies were picked and plated in 24 well SNL feeders’ plates for expansion and characterization. For embryoid bodies (EB) generation, EPSC lines were harvested and feeders removal was performed in T25 flasks for 30 minutes at 37 degrees to allow feeders to attach to the bottom and floating EPSC harvested were transferred in ultra-low attachment 96 well plates at different densities (60K, 45K, 35K and 20K) in EB media (DMEM/Knockout Invitrogen #10829-018, Fetal Bovine serum Gibco #16141061, non-essential amino acid Invitrogen #11140, glut-pen-strep Invitrogen #10378 and β mercapto-ethanol) changed every other days. After 7 days, they were transferred in 24 well plates on gelatine coated glass coverslips and after 10 to 14 days, cells were fixed with PFA 4%, 30 minutes at room temperature and immunocytochemistry was performed. EPSC were induced into iNSC following two protocols. The Gibco protocol (iNSCGibco) previously described and a bespoke protocol. For the latter, feeders were removed and EPSC colonies dissociated with accutase and plated in geltrex coated 6 well plates in EPSCM and rock inhibitor 10μM at density from 0.25 to 0.5x106 cells/well, media was changed the next day with N2B27 induction media (Neurobasal medium Gibco #12348017, DMEM/F21 Gibco #11330032, N2 supplement #17502048, B27 supplement 12587010, EGF and bFGF 10ng/ml peprotech and Pen-Strep-Glut Sigma) and afterwards every 4 days for a week. Cells were passaged with accutase and plated at density of 0.5x106 cells/well in geltrex coated 6 well plates and media was replaced by N2 expansion medium (DMEM/F21 Gibco #11330032, N2 supplement #17502048, EGF, bFDF and Pen-Strep-Glut) and changed every 2 days, passages were performed by enzymatic cells dissociation with accutase 2 to 3 times to establish iNSC N2B27 lines (iNSCN2B27). eNSC, iNSCGibco and iNSCN2B27 were frozen in synth-a-Freeze cryopreservation medium (Gibco #A12542).
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| Growth protocol |
Primary endogenous NSC (eNSC) were isolated from the sub ventricular zone (SVZ) of 3 months old C57Black6/J mice following established protocols. Briefly, SVZ were dissected from the cerebral hemispheres and digested using Papain dissociation kit (PDS, Worthington #0031150 and #003153), plated at a density of 4x104 mL-1 and grown as Neurospheres (NS) for 4 days in DMEM/F12 (Invitrogen #31330), mouse recombinant EGF and human recombinant FGF (20ng/ml, Peprotech #31509 and #100-18B), 2% B27 (Invitrogen #12587) and Pen/Strep 1X (Invitrogen #15140), then media was changed every 4 days. After the first passage, cells were plated in adherent condition in geltrex (Gibco # A1413302) coated 6 well plates (corning # 3516) at a density of 0,5x10^6 cells. Dissociation between passages was performed enzymatically with accutase (Millipore #SCR005). After 2 passages, media was changed to neural expansion media (Gibco Advanced DMEM/F 12 0,5X #12634, Neurobasal medium 0,5X # 21103, 1% Pen-Strep and 2% neural induction supplement #A1647801). Dura mater was collected from C57Black6/J mice before dissection of the hemispheres for eNSC isolation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA and DNA were extracted from cell pellets using the RNA/DNA/Protein Purification Plus kit (NORGEN, #47700), following the manufactured protocol.
RNA samples were sequenced on HiSeq4000, at 75 bp PE after polyA selection. For the RRBS library preparation, DNA samples were digested overnight with restriction enzyme Mssp1 (Agilent), then prepared following the manufactured protocol of NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (Biolab, E7645). Finally, bisulfite conversion following Zimo kit (Qiagen, D5001) run on NextSeq 500 Mid Output Run 150 cycles.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
GC-CV-7163-6_S6
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| Data processing |
RRBS-Seq quality and alignment: Quality was assessed via FastQC. Trimming of low quality reads and adapters was performed via TrimGalore v. 0.4.5-1 with “--rrbs” mode enabled. Subsequently, Bismark v. 0.22.1 was used for the alignment of reads, calling the subroutines “nucleotide-coverage” and “methylation-extractor”. Default settings were used, including the usage of Bowtie2 as background aligner.
RRBS-Seq post-processing: The coverage files were post-processed via edgeR. CpG sites with a total coverage – i.e. the sum of methylated (Me) and unmethylated (Un) reads – greater than 10 across all samples were retained as well as only those in chromosomes 1-19 and X. M-values were calculated as log2(Me+2)-log2(Un+2).
RNA-Seq quality and alignment: Quality was assessed via FastQC and trimming of low quality reads and adapters was performed via TrimGalore v. 0.4.5-1 with default parameters. The pseudoalignment package Salmon v. 0.13.1 was used to obtain transcript per million (TPM) expression levels, normalized for library size and transcript length.
RNA-Seq post-processing: Transcript to gene quantification was performed via biomaRt in R.
Genome_build: Ensembl GRCm38
Supplementary_files_format_and_content: RRBS-Seq: coverage files (cov.gz); RNA-Seq: TPM count table (csv)
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| Submission date |
Jul 13, 2020 |
| Last update date |
Sep 17, 2021 |
| Contact name |
Silvia Marino |
| E-mail(s) |
s.marino@qmul.ac.uk
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| Organization name |
Queen Mary University of London
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| Street address |
4, Newark St
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| City |
London |
| State/province |
- |
| ZIP/Postal code |
E12AT |
| Country |
United Kingdom |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE154367 |
Comparative analysis of glioblastoma initiating cells and patient-matched EPSC-derived neural stem cells as a discovery tool and drug matching strategy [Seq] |
| GSE155994 |
Comparative analysis of glioblastoma initiating cells and patient-matched EPSC-derived neural stem cells as a discovery tool and drug matching strategy |
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| Relations |
| BioSample |
SAMN15520491 |
| SRA |
SRX8722973 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4669696_GC-CV-7163-6_S6_pe.bismark.cov.gz |
64.1 Mb |
(ftp)(http) |
COV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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