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Sample GSM2938848

Query DataSets for GSM2938848

Status

Public on Jan 09, 2019

Title

RNA-seq_dopaminergic neuron sample 1

Sample type

SRA

Source name

dopaminergic neurons from 14.5 day embryo

Organism

Mus musculus

Characteristics

genotype: TH-GFP
tissue: dopaminergic neuron
developmental stage: embryo
age (day): 14.5

Growth protocol

All procedures were conducted in compliance with the European and French legislations (EU directive 2010/63/UE), and were approved by a committee for ethical experiments (A751319). To purify dopaminergic (DA) neurons by fluorescence-activated cell sorting (FACS) prior to RNA-seq and ATAC-seq, we used TH-GFP mice, in which GFP is expressed under the control of the Th promoter40. This transgenic line, maintained on C57BL/6J background, was generously given by H. Okano. To isolate serotonergic (5-HT) neurons by FACS, we were generously given by C. Parras Mash1-CRE x ROSA YFP mice, in which YFP is expressed in 5-HT neurons43. Mice had ad libitum access to food and water, and were housed in cages containing up to 5 animals under temperature-controlled conditions and maintained on a 12:12 hours light/dark cycle. To obtain E14.5 embryos, males from these transgenic lines were mated with Swiss wild-type females overnight, and pregnancies confirmed the next morning by inspection of the vaginal plug, defining embryonic day 0.5.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from DA or 5-HT neurons using an RNeasy Microkit (Qiagen) following manufacturer´s instructions. RNA was treated with DNAse I (Qiagen) for 20 minutes at room temperature to prevent genomic DNA contamination. For RT-qPCR, RNA concentrations were determined by spectrophotometry (Nanodrop 2000c, THERMO Scientific). For RNA-seq, a High Sensitivity RNA ScreenTAPE analyzer (Agilent Technologies) was used to assess RNA concentrations as well as the RNA integrity number (RIN) to verify RNA quality for all tested samples. RNA was stored at−80 °C until reverse transcription or RNA-Seq.
Stranded library was prepared using TotalScript RNA sequencing kit (Epicentre) following manufacturer’s recommendation. For purified samples of 5HT neurons, non-stranded library were prepared using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech) following manufacturer’s recommendation. 3 DA and 3 5-HT libraries were sequenced using NextSeq500 HighOutputKit v2 (300cycles) cartridge (FC-404-2004 Illumina). Sorted cells by FACS were collected in Neurobasal medium with B27 supplement (Life Technologies), 2% FBS and kept at 4°C until ATAC-Seq. Cells were centrifuged at 500 g, at 4°C during 20 min. Cells were resuspended in 25 μl of lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) during 10 min at 4°C. Then supernatant was taken out after a centrifugation at 500 g, at 4°C during 30 min. For transposase reaction, the pellet was resuspended in 25 μl of 12.5 μl 2x TN buffer; 2 μl of Tn5; 10.5 μl d’H2O and incubated at 37°C for 1 h. Then 5 μl of clean-up buffer (900 mM NaCl, 300 mM EDTA, 5% SDS) were added with 2 μl of 5% SDS and 2 μl of Proteinase K, and cells were incubated for 30 min at 40°C. Samples were then cleaned with two SPRI clean up (Agencourt © AMPure ©XP), with 68 μl of SPRI beads, eluted in 13 μl of buffer EB (Qiagen Cat No./ID: 19086). Extracted DNA concentration was measured by ScreenTAPE analyzer (Agilent Technologies). To generate libraries, PCR reactions were performed using the kapa PCR mix (Kapa biosystem) with 12.5 μl Kapa, 1 μl primers and 11.5 μl of sample, with the NextEra primers (1 μl /primer). PCR conditions were performed as described: 98°C during 2 min and then 9 cycles of 98°C during 20 s, 63°C during 30 s, 72°C during 1 min. Then, a new SPRI clean-up was made to do a size cut off of amplified PCR products and after that, DNA concentration was measured by ScreenTAPE analyzer (Agilent Technologies). Finally, a second PCR was performed with the same conditions as the first one and a last SPRI clean-up was made and library of tagged open chromatin was ready to be sequenced.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

lncRNA
sample name: ARN13avril15_1
DA-1
DA_FPKM_allTranscripts.xlsx

Data processing

Steps for quality control were identical to those used for RNA-seq data treatment (Trimmomatic, FastQC). Paired-end reads were mapped to the mouse genome (build mm9) with Bowtie2. Duplicate reads were discarded with the Picard tools. Peaks were called using the MACS2 program with the option callpeak. Individual peaks separated by less than 100 bp were merged with BEDOPS and features annotations were obtained from the HOMER mm9 database. Data from ATAC-seq and RNA-seq results were intersected based on overlaps between a given ATAC peak and the first/last nucleotide of a TSS/TTS, respectively.
Genome_build: mm9 (mouse)
Supplementary_files_format_and_content: excel files containing FPKM for each 5HT and DA sample
Supplementary_files_format_and_content: ATAC-seq BED file (6+3 BED file see http://genome.ucsc.edu/FAQ/FAQformat.html#format13) with peak

Submission date

Jan 17, 2018

Last update date

Jan 09, 2019

Contact name

Philippe Ravassard

E-mail(s)

philippe.ravassard@icm-institute.org

Organization name

Brain & Spine Institute

Street address

47, boulevard de l'Hôpital