Brain_PND1_Het_9
Mus musculus
Protocols: Mouse tissues were collected on PND1 and PND5. Mice were sacrificed with CO2 asphyxiation and organs/tissues were rinsed in saline, snap-frozen in liquid N2 and stored in -80°C until processing. For extraction of RNA samples, tissues were pulverized in liquid N2 with a mortar and pestle and total RNA was purified with RNeasy Mini Kit and treated with RNase-free Dnase I. RNA purity, integrity and concentration were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc, USA). Only RNA with a RIN value of 8.0 or higher and a ratio (28s/18s) of above 1.8 was taken forward for sample preparation. Samples were processed for library constructions following the manufactory instructions (Illumina Tru Seq Stranded Total RNA sample preparation v2 Guide, Part #15031048 Rev.E October 2013 - “Low sample protocol”)*. In brief, 0.5 µg of total RNA from each tissue were depleted for cytoplasmic rRNA using the Ribo-Zero ribosomal reduction chemistry, chemically fragmented for 8 min at 94oC, and processed for 1st strand synthesis cDNA and then 2nd strand synthesis using dUTP instead of dTTP. After some purification steps, the cDNA was then end-repaired, purified, adenylated at the 3'-ends, and purified before adding the indexed adaptor sequences using the TruSeq Stranded LT Kit Index set A. Each library preparation was then enriched by 10 cycles of PCR, purified and finally validated in regards to size and concentration. For sizing, libraries were analyzed on the Agilent 2100 Bioanalyzer using a DNA 1000 kit from Agilent Technologies. The Libraries were quantified by qPCR using the KaPa Library quantification Kits (KaPa Biosystems, Cat KK4824). Samples were pooled in sets of 12 libraries, and a final concentration of 16 pM denatured library was used for paired-end deep sequencing using an Illumina HiSeq1500.
ENA-FIRST-PUBLIC
2016-08-25T17:01:15Z
ENA-LAST-UPDATE
2018-03-09T02:41:25Z
External Id
SAMEA3463406
INSDC center name
University of Southern Denmark Department of Biochemistry and Molecular Biology
INSDC first public
2016-08-25T17:01:15Z
INSDC last update
2018-03-09T02:41:25Z
INSDC status
public
Submitter Id
E-MTAB-3664:Brain_PND1_Het_9
age
1
broker name
ArrayExpress
common name
house mouse
genotype
heterozygous
organism part
brain
sample name
E-MTAB-3664:Brain_PND1_Het_9
scientific_name
Mus musculus
sex
male