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Sample GSM3293749

Query DataSets for GSM3293749

Status

Public on Dec 25, 2018

Title

VM2_12.5

Sample type

SRA

Source name

ventral midbrain

Organism

Mus musculus

Characteristics

tissue: ventral midbrain
developmental stage: E12.5
genotype: TH-GFP

Growth protocol

Mouse embryos were obtained from TH-GFP animals (Matsushita et al, J Neurochem. 82: 295, 2002) that were mated overnight, and noon of the plug day was considered E0.5. Embryos were dissected out of the uterine horns at E11.5 - E14.5 and placed in ice cold sterile PBS where brain regions were dissected under a stereomicroscope with a UV attachment to detect GFP. VM samples corresponded to domains M3 to M7 of the floor and basal plate (Nakatani et al, Development 134: 2783, 2007). Tissue samples were collected in separate tubes and stored at - 80 ̊C until RNA isolation.

Extracted molecule

total RNA

Extraction protocol

Total RNA isolation was performed with the RNeasy kit (Qiagen). The RNA integrity and concentration was checked using Qubit and 2200 TapeStation (Agilent).
Illumina TruSeq libraries were prepared using the kit and standard protocol from Illumina.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

E12dot5_TruSeq_mm10_UCSC_20140930_153655_TS23

Data processing

Read quality filtering: Every read that was considered valid by the Illumina control software was processed and filtered as follows: a) any 3' bases with a quality score of 'B' were removed; b) if the read ended in a poly(A)-sequence leaving less than 25 transcript-derived bases, the read was discarded; c) if the remaining sequence consisted of less than six non-A bases, or a dinucleotide repeat with less than six other bases at either end, the read was discarded.
Alignment:the reads were aligned to the genome using the Bowtie aligner, allowing for up to three mismatches and up to 24 alternative mappings for each read. Any reads with no alignments were re-aligned against an artifical chromosome, containing all possible splice junctions arising from the exons defined by the known transcript variants. Reads mapping within these splice junctions were translated back to the corresponding actual genomic positions. The UCSC transcript models were used for the expression level calculation. If a locus had several transcript variants, the exons of these were merged to a combined model that represented all expression from the locus. To account for incomplete cap site knowledge, the 5' ends of all models were extended by 100 bases, but not beyond the 3' end of any upstream nearby exon of another gene of the same orientation.
Annotation and quantitation: Reads that had one or more repeat mappings that was outside exons, was assigned randomly as one of these repeats and contributed to the summarized read count of that repeat class. Else, if it had one or more mappings to exons, it was assigned randomly to one of the exons, even if the sequence was repeat-like. If it had no exon mapping, it was assigned randomly at one of the mappings. The expression level of each transcript model was taken as the total number of reads at all its possible mapping positions, normalized as Reads Per Kilobase mRNA Per Million (RPKM).
Genome_build: UCSC mm10
Supplementary_files_format_and_content: Tab-separated file with RPKM.

Submission date

Jul 19, 2018

Last update date

Dec 25, 2018

Contact name

Sten Linnarsson

Organization name

Karolinska Institutet

Department

Medical Biochemistry and Biophysics

Lab

Molecular Neurobiology