Ovation® RNA Amplification System V2

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Always keep thawed reagents and reaction tubes on ice	do not use DEPC-treated water, only water provided in the kit or nuclease-free water

Upstream tips
Always keep thawed reagents and reaction tubes on ice
Protocol tips
do not use DEPC-treated water, only water provided in the kit or nuclease-free water

Publication protocol

Tissue dissection, FACS, RNA amplification, and Microarray and RNA sequencing
Preparation of RNA sequencing samples was performed as described previously26. P3 to P7 mouse cochleae from Gad1-GFP mice for the purification of SCs in the GER region and PV-Cre;Ai6 mice for the purification of HCs and SGNs were dissected out. SCs are positive for GFP in the Gad1-GFP line, and HCs and SGNs are positive for GFP in PV-Cre;Ai6 mice. The GFP-labeled HC and SGN regions were mechanically separated during the cochlear dissection as there is a clear structural separation between the two regions. The isolated tissues were treated with activated papain (20U/ml; Worthington) for 20 min followed by 2 min crude typsin (0.5 mg/ml, Sigma-Aldrich) to achieve complete dissociation. Then, fluorescence-activated cell sorting (FACS) was performed at the Flow Cytometry Core Facility of USC. Cell suspensions were fed into a BDAriaII sorter and purified using 488 nm laser excitation and a 100-μm cytoNozzle. 2000 cells for each distinct cell population were collected into DMEM plus 10% FBS and pelleted down through centrifuge. RNA was extracted from the collected cells using PicoPure RNA isolation kit (Arcturus). Each RNA sample was then amplified using WT-Ovation Pico amplification kit (Nugen) and sequenced with Illumina HiSeq 2000 (Illumina) at the USC Genomics Center following the manufacturer’s instructions. Microarray samples were prepared from P5 to P7 mouse cochleae as described previously23. HCs were purified with FASC after staining wildtype cochleae with 5 μM FM1-43 (Invitrogen) or after dissecting tdTomato-labelled HC regions from PV-Cre;Ai14 (with SG trimmed off) mice. There were no differences in fluorescence expression pattern in the cochlea between PV-Cre;Ai14 and PV-Cre;Ai6 animals. For the SGN population, fluorescent SGNs from PV-Cre;Ai14 cochleae, in which the HC region was mechanically trimmed off, were collected. Preparation of RNA samples was similar to that for the RNA-sequencing experiments.

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Manufacturer protocol

Download the product protocol from NuGEN for Ovation® RNA Amplification System V2 below.

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