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Sample GSM6685258 Query DataSets for GSM6685258
Status Public on Dec 01, 2022
Title iNeuron_TDGKD_day17_RNAseq_rep6
Sample type SRA
 
Source name iNeuron
Organism Homo sapiens
Characteristics harvested day: day17
genotype: TDG KD
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.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model NextSeq 550
 
Description iNeuron-TDGKD-timeCouse-RNA-seq.count.txt
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.
 
Submission date Oct 26, 2022
Last update date Dec 01, 2022
Contact name Wei Wu
Organization name National Cancer Institute
Department Center for Cancer Research
Lab Laboratory of Genome Integrity
Street address 9000 Rockville Pike
City Bethesda
State/province MD
ZIP/Postal code 20892
Country USA
 
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
Relations
BioSample SAMN31467524
SRA SRX18039119

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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