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Sample GSM4007738

Query DataSets for GSM4007738

Status

Public on May 01, 2020

Title

mmRNAldP00R2

Sample type

SRA

Source name

Retina

Organism

Mus musculus

Characteristics

tissue: Retina
genotype: GlastCreERT2/Sun1-sGFP, CD1
condition: Light damage
cell population of facs sorting: GFP+
time: 0hr
replicate: Replicate 2

Treatment protocol

For NMDA damage, adult mice, either CD1 or GLASTCreERT2; Sun1-sGFP mice at ~2 months of age, were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt-ended needle. Mice were sacrificed, and retinas were collected at indicated timepoints. For light damage, the mice were reared in cyclic 12-hour low light/12-hour dark conditions at the University of Florida animal housing facility. Prior to light damage, mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter (Thermo Fisher Scientific Inc., Waltham, MA) and set to 2000 lux. To induce death of rods and cones, adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days, then transferred to clear polycarbonate tanks placed between four fluorescent bulbs (20,000 lux) and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2-phenoxyethanol in system water. To induce amacrine and ganglion cell death, we injected N-methyl-D-aspartic acid (NMDA) (Sigma-Aldrich; St. Louis, MO) intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2-phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2-phenoxyethanol in system water.

Growth protocol

To induce Cre recombination, GLASTCreERT2; Sun1-sGFP mice at ~2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for 2 consecutive days at 1mg/dose. Nfialox/lox; Nfiblox/lox, and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1-sGFP mice. To generate MG-specific loss of function mutants of NFIa/b/x genes, 3 week-old GLASTCreERT2; NFIa/bxlox/lox;Sun1-sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate-controlled pathogen free facility on a 14 h-10 h light/dark cycle (07:00 lights on – 19:00 lights off). The adult zebrafish used in these studies were 6 to 12 months old (3-5cm in length). Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted from both GFP-positive and GFP-negative cell fractions using miRNAeasy Mini Kit (#217004, Qiagen). Ribosomal RNA was depleted, and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo-ZeroTM Gold (# 15032619, Illumina). For tagmentation, cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler (Illumnia Nextera DNA library prep kit, #FC1211030). DNA was cleaned up using MinElute PCR purification kit (#28006, Qiagen) and eluted in 10UL of EB buffer.
Flow-sorted RNA samples were sent to the Deep Sequencing and Microarray Core (Johns Hopkins University) for library preparation and sequencing. Around 8-10 libraries were pooled and sequenced for paired-end 75 cycles using the NextSeq 500 system with ~400-500 million reads per run, resulting in ~45-55 million reads per library. Sorted cells (~50k-75k) from GFP-positive samples were used for ATAC library preparation. Completed ATAC-Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired-end 75 cycles using the NextSeq 500 system with ~400-500 million reads per run.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR
Raw counts of genes were further used to calculate FPKM (Fragments Per Kilobase Of Exon Per Million) and identify differentially expressed genes through EdgeR
After removing adaptors using cutadapt (58), 50bp paired-end ATAC-Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters
We filtered reads from chromosome M and Y, and included high mapping-quality reads (MAPQ score > 10) through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program.
ATAC-Seq peak regions were called using MACS2 with parameters --nomodel --shift -100 --extsize 200. The blacklisted regions in mouse were excluded from peak regions (https://www.encodeproject.org/annotations/ENCSR636HFF/).
We counted the raw fragments for each peak region using HTSeq.
Genome_build: GRCm38/mm10 and GRCz10/danRer10
Supplementary_files_format_and_content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome, start, stop, name, length of peak region, strand, integer score for display, fold-change, -log10(pvalue), -log10(qvalue), relative summit position to peak start.

Submission date

Aug 05, 2019

Last update date

May 01, 2020

Contact name

Jie Wang

E-mail(s)

jwang240@jhmi.edu

Organization name

Johns Hopkins University

Department

Ophthalmology