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Sample GSM4753635

Query DataSets for GSM4753635

Status

Public on Jul 02, 2021

Title

rRPE1

Sample type

SRA

Source name

E4 RPE 6 hours post retinectomy and FGF2 treatment (reprogrammed RPE; rRPE)

Organism

Gallus gallus

Characteristics

developmental stage: Embyonic day 4
tissue: retinal pigment epithelium (RPE)

Extracted molecule

polyA RNA

Extraction protocol

Embryos were collected and infiltrated at 4°C with a sucrose gradient followed by 2:1 25% sucrose:OCT compound (Sakura Finetek, Torrance, CA) for 1 h and snap frozen in dry ice and methylbutane. Cryosections (12μm) were collected on PEN Membrane Frame Slides (ThermoFisher Scientific) followed by fixation in 70% ethanol at -20°C for 30 sec. Slides were rehydrated in 4°C DEPC-treated water for 30s, stained with hematoxylin (Sigma, St. Louis, MO) for 10 sec, and dehydrated in a 70%, 95%, and 100% ethanol gradient for 30 seconds each. LCM was performed using a Veritas Laser Capture Microdissection system and software to collect RNA samples for library preparation. RPE was isolated from sections using IR capture and UV dissection on CapSure HS LCM Caps (ThermoFisher Scientific, Waltham, MA), and total RNA extraction was performed using PicoPure RNA Isolation Kit (Arcturus, Applied Biosystems, Foster city, CA), including a treatment with DNase I.
Total RNA was processed for library construction by Cofactor Genomics (http://cofactorgenomics.com, St. Louis, MO) according to the following procedure. Briefly, total RNA was reverse-transcribed using an Oligo(dT) primer, and limited cDNA amplification was performed using the SMARTer® Ultra® Low Input RNA Kit for Sequencing – v4 (Takara Bio USA, Inc., Mountain View, CA ). The resulting full-length cDNA was fragmented and tagged, followed by limited PCR enrichment to generate the final cDNA sequencing library (Nextera® XT DNA Library Prep, Illumina, San Diego, CA). Libraries were sequenced as single-end 75 base pair reads on an Illumina NextSeq500 following the manufacturer's instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Read trimming with Trim Galore v0.6.1 and Cutadapt v1.18
Alignment to GRCg6a with STAR v2.7.3
Transcript quantification with Stringtie v2.0.4 and Ensembl 98
Gene count normalization with DESeq2 v1.22.2
Genome_build: GRCg6a
Supplementary_files_format_and_content: DESeq2 normalized gene counts

Submission date

Aug 29, 2020

Last update date

Jul 02, 2021

Contact name

Katia Del Rio-Tsonis

E-mail(s)

delriok@miamioh.edu

Organization name

Miami University

Department

Biology