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| Status |
Public on Dec 01, 2022 |
| Title |
iNeuron_WT_day30_RNAseq_rep2 |
| Sample type |
SRA |
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| Source name |
iNeuron
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| Organism |
Homo sapiens |
| Characteristics |
harvested day: day30
genotype: WT
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| Growth protocol |
For neuronal differentiation, 20–25 million iPSCs were plated on day 0 onto a 15-cm plate in N2 medium (knockout Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium; Life Technologies Corporation, cat. no. 12660012) with N2 supplement (Life Technologies, cat. no. 17502048), 1× GlutaMAX (Thermofisher Scientific, cat. no. 35050061), 1× MEM nonessential amino acids (NEAA) (Thermofisher Scientific, cat. no. 11140050), 10 μM ROCK inhibitor (Y-27632; Selleckchem, cat. no. S1049), and 2 μg/ml doxycycline (Clontech, cat. no. 631311). N2 medium was changed once a day for two more days. On day 3, cells were replated onto freshly prepared dishes coated with freshly prepared poly-L-ornithine (PLO; 0.1 mg/ml; Sigma, cat. no. P3655-10MG). Pre-neuron cells were cultured in i3Neuron Culture Media: BrainPhys media (Stemcell Technologies, cat. no. 05790) supplemented with 1× B27 Plus Supplement (ThermoFisher Scientific, cat. no. A3582801), 10 ng ml−1 BDNF (PeproTech, cat. no. 450-02), 10 ng ml−1 NT-3 (PeproTech, cat. no. 450-03), 1 μg ml−1 mouse laminin (Sigma, cat. no. L2020-1MG), and 2 μg ml−1 doxycycline (Clontech, cat. no. 631311). i3Neurons were then fed by half media change on day 6 and then, collected on day 7. For iMacrophage transdifferentiation, 10 million C10 cells were induced to reprogram by the addition of 100 nM of β-estradiol (Sigma-Aldrich; Cat. #E8875) and grown with 10 ng/ml of IL-3 (Peprotech; Cat. #213-13), CSF-1 (Peprotech; Cat. #315-02) and 350 nM Ascorbic acid (Sigma-Aldrich; Cat. # A8960) and then collected on day 1, day 2 and day3.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
To extract RNA, cells were plated on 6-well dishes, washed with PBS, and then 500 L of tri-reagent (Zymo research corporation, Cat. No. R2050-1-200) was added directly to the cells. The lysed cells were collected using a cell scraper. To isolate RNA, we used a Direct-zol RNA miniprep kit (Zymo Research Corporation, Cat. No. R2052), following manufacturer’s instructions including the optional DNAse step.For RNA sequencing, two biological replicates were sequenced. Total RNA was enriched for polyA and sequenced 2x75 bp on a NextSeq550 machine.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
iNeuron-TDGKD-timeCouse-RNA-seq.count.txt
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| Data processing |
SAR-seq, END-seq and SEAL reads were aligned to the reference genome (hg19 for human i3Neuron and mm10 for mouse macrophage cells using bowtie (v1.1.2) with parameters -n 3 -l 50 -k 1 for END-seq and -n 2 -l 50 -m 1 for the rest. RNA-seq reads were aligned by STAR (v2.7.6a). PB-seq was mapped by BSMAP (v2.90) with parameters -p 10 -w 2 -v 0 -q 30. Functions “view” and “sort” of samtools (v1.11) were used to convert and sort the aligned sam files to sorted bam files. Bam files were further converted to bed files by the bedtools (v2.29.2) bamToBed command. Mitochondrial reads were removed in SAR-seq for intensity comparisons.
For RNA-seq, fragments per kilobase of Read per million mapped reads (RPKM) was calculated by cufflinks based on the annotation from GENCODE v33 for iNeuron. And count was used for iMacrophage using GENCODEvM24.
Assembly: hg19 for human, mm10 for mouse.
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| Submission date |
Oct 26, 2022 |
| Last update date |
Dec 01, 2022 |
| Contact name |
Wei Wu |
| Organization name |
Center for Excellence in Molecular Cell Science
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| Department |
Center for Excellence in Molecular Cell Science
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| Street address |
320 yueyang road
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| City |
Shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
200031 |
| Country |
China |
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| Platform ID |
GPL21697 |
| Series (2) |
| GSE210316 |
Active DNA demethylation promotes cell fate specification and the DNA damage response [RNA-Seq] |
| GSE210317 |
Active DNA demethylation promotes cell fate specification and the DNA damage response |
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| Relations |
| BioSample |
SAMN31467522 |
| SRA |
SRX18039121 |
| 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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