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| Status |
Public on Aug 11, 2023 |
| Title |
RL416: Day_13_Primed_32F |
| Sample type |
SRA |
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| Source name |
Human reprogramming intermediate cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Human reprogramming intermediate cells
treatment: Day_13_Primed
background: 32F
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| Treatment protocol |
On day 8, the cells were switched to primed, naive, naive-to-primed (NtP) or transient-naive-treatment (TNT) reprogramming protocols. For primed: Cells were transient primed essential8 (E8) culture media after the initial 7 days culturing in fibroblast media. For Naive: Naive hiPSCs colonies were established through extended naive culturing. For TNT: cells were transient naive culture for 5 days after the initial 7 days culturing in fibroblast media, followed by culturing in primed E8 media. For NtP: Naive hiPSCs colonies were established through extended naive culturing and then transitioned the cells to a primed pluripotent state in E8 media to give rise to naive-to-primed hiPSCs.
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| Growth protocol |
To generate human primed and naïve iPSCs used for this study, somatic cell reprogramming with the Cytotune 2.0 kit, experiments were performed according to the manufacturer’s instructions (Invitrogen). Briefly, human dermal fibroblast (adult) were seeded at ~5x104 cells per well of a 6-well plate in fibroblast medium containing DMEM (Gibco), 10% FBS (Hyclone), 1% nonessential amino acids (Gibco), 1mM GlutaMAX (Gibco), Pen-strep (Gibco), 0.1mM 2-mercaptoethanol (Gibco) and 1mM sodium pyruvate (Gibco) . After 2 days, cells were transduced with Sendai viruses in fibroblast medium with multiplicity of infections (MOIs) as follows, KOS MOI=5, c-MYC MOI=15, KLF4 MOI=6. After 24 hours, the medium was replaced with fresh fibroblast medium and media were changed every other day thereafter. On day 7, cells were trypsinized and seeded onto a layer of iMEF feeders at ~0.5x105~1x105 per flask in fibroblast medium. The next day, the cells were switched to primed or naive media. 21-24 days after transduction, cells were passaged and expanded. Reprogramming intermediates were collected at the indicated time and isolated by FACS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated with the Qiagen Blood and Tissue Kit according to manufacturer's instructions. 0.5% (w/w) of unmethylated lambda phage DNA (Promega) was added to the sample genomic DNA for the purpose of an unmethylated control to measure the bisulfite non-conversion frequency in each sample.
Genomic DNA was fragmented with a Covaris S2 sonicator to a mean length of 200 bp, then end-repaired, A-tailed, ligated to methylated Illumina TruSeq adapters, and subjected to PCR amplification with KAPA HiFi Uracil+ DNA polymerase (KAPA Biosystems). Single-end sequencing was performed on a HiSeq1500, NextSeq500 or NovaSeq.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Sequencing adapters were trimmed with BBduk with the options mink = 3, qtrim = r, trimq = 10 minlength = 20 before alignment to hg19 with Bowtie and BSseeker2 with the option -n 1. PCR duplicates were removed using Sambamba and DNA methylation levels at base resolution calculated using CGmap tools. The non–conversion rate was calculated using the DNA methylation levels for the spiked–in lambda phage genome. When DNA methylation levels were calculated for regions such as promoters, enhancers, DMRs or ICRs, the methylation level was calculated as a coverage–weighted average by summing the number of methylated C calls (mCG) and dividing that by the total number of reads with either a C or T call (CG). This is represented in the figures and text as mCG/CG. To calculate methylation in CH contexts (where H = A, T or C), the level of methylation was calculated as above (mCH/CH) with the non–conversion rate subtracted from this value. When CH methylation was calculated for individual contexts, for example CA methylation, the non–conversion rate for that context was subtracted from the calculated methylation levels. For CA methylation browser tracks, mCA/CA was calculated for 5kb sliding windows (1kb slide), with the CA methylation non–conversion rate for that library subtracted from each window. To calculate per-read methylation, reads classified as methylated had methylation calls at every CG position in the read; unmethylated reads had zero methylation calls at CG positions; partially methylated reads had at least one CG methylation call and one non-methylated CG call.
Genome_build: hg19
Supplementary_files_format_and_content: CGmap file generated with CGmap tools. File has 8 columns: [1]chromosome [2] nucleotide on Watson (+) strand [3] position [4] context (CG/CHG/CHH) [5] dinucleotide-context (CA/CC/CG/CT) [6] methylation-level = #_of_C / (#_of_C + #_of_T) [7] #_of_C (methylated C, the count of reads showing C here) [8] = #_of_C + #_of_T (all Cytosines, the count of reads showing C or T here).
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| Submission date |
Nov 05, 2020 |
| Last update date |
Aug 11, 2023 |
| Contact name |
Ryan Lister |
| E-mail(s) |
ryanlister@gmail.com
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| Phone |
61864884407
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| Organization name |
The University of Western Australia
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| Street address |
35 Stirling Highway
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| City |
Perth |
| State/province |
WA |
| ZIP/Postal code |
6009 |
| Country |
Australia |
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| Platform ID |
GPL18460 |
| Series (1) |
| GSE160933 |
Transient naive reprogramming corrects hiPS cells functionally and epigenetically [WGBS 2] |
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| Relations |
| BioSample |
SAMN16676906 |
| SRA |
SRX9444985 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4886708_RL416_D13_32F_Primed.rmdup.CGmap.gz |
4.6 Gb |
(ftp)(http) |
CGMAP |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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