Computational protocol: Deletion of lynx1 reduces the function of α6* nicotinic receptors

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Protocol publication

[…] For laser capture microdissection, C57BL6/DBA WT mice were deeply anesthetized with sodium pentobarbital (100 mg/kg; i.p.) and sacrificed by decapitation. Our methods pipeline for fabricating cDNA libraries via laser capture microdissection, and performing RNA-Seq were previously described []. Briefly, whole brains from 4 month old male mice (n = 3) were collected (post-mortem interval of < 5 min), fresh frozen over dry ice, and stored at -80°C. Midbrain cryostat [] sections (20 μm) were mounted on UV-treated Zeiss Membrane Slides (1.0 PEN NF), air dried for 5 minutes, and stained with cresyl violet for 1 minute. The sections were rinsed, dried and then visualized under brightfield illumination at 400X magnification on a Zeiss PALM Laser Capture Micro Dissection microscope. Twenty putative DA+ cell bodies from the SNc were dissected using multiple low laser energy pulses, and were catapulted into Zeiss 200 μL adhesive caps. Cell lysis solution (Illumina, San Diego, CA) containing 3′ SMART reverse transcription primers and quantitation controls (“spikes”) were then added into the pool of cells prior to freezing.To fabricate cDNA libraries, we prepared amplified cDNA from RNA, using Clontech's SMARTer™ Ultra Low RNA system for Illumina Sequencing (Clontech, Mountain View, CA) as previously described []. Poly(A)+ RNA was reverse transcribed through oligo dT priming to generate full-length cDNA, which was then amplified using 22 cycles, using Clontech's Advantage 2 PCR system. RNA-Seq libraries were constructed using the Nextera DNA Sample Prep kit (Illumina). cDNA was “tagmentated” at 55°C with Nextera transposase, and tagmented DNA was purified using Agencourt AMPure XP beads (Beckman Coulter Genomics). Purified DNA was amplified using five cycles of Nextera PCR. After quality control measures of yield and fragment length distribution were taken using the Qubit fluorometer (Invitrogen, Carlsbad, CA) and the Agilent (Santa Clara, CA) Bioanalyzer, 50 bp or 100 bp sequencing reads were generated on the Illumina HiSeq instrument. Each sequencing library generated > 20 million uniquely mapping reads.For computational analysis, 50 bp or 100 bp sequence tags were mapped to the mouse genome using TopHat 1.3.2 []. We quantified transcript abundance (FPKM: fragments per kilobase per million mapped reads (expression values)) using Cufflinks. We annotated the transcripts with genome annotations provided by ENSEMBL. Data were analyzed and graphs were generated using GraphPad Prism 5. […]

Pipeline specifications

Software tools TopHat, Cufflinks
Application RNA-seq analysis
Chemicals Acetylcholine, Dopamine, Nicotine, Rubidium