title: GSM2258080: Shock30; Mus musculus; RNA-Seq
library_construction_protocol: Manual cell sorting and RNA sequencing. Manual sorting of fluorescent cells was carried out as first described in24. Briefly, Arc-dVenus transgenic male C57BL/6 mice were sacrificed under isoflurane anesthesia 6hrs after behavioral procedures and their brains were quickly removed and transferred into ice-cold oxygenated ACSF, containing 126 mM NaCl, 20 mM NaHCO3, 20 mM dextrose, 3 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, 2 mM MgCl2, 50 mM APV, 20 mM DNQX, 100 nM TTX. Acute 330 μm coronal brain slices were prepared and incubated in a protease E (1.2 mg/mL) (Sigma) containing oxygenated ACSF for 50min. After 15min of washes in the ACSF, TeA tissue was microdissected using a pair of fine scissors under a fluorescent dissecting microscope (Leica M165FC). The dissected TeA tissue was then triturated in ACSF using a series of three Pasteur pipettes of decreasing tip diameters, and the dissociated cells were transferred into a small petri dish. With visual control under a fluorescent dissecting microscope, Venus+ neurons were aspirated into a micropipette with a 30–50 μm tip diameter and transferred into a clean petri dish. A total of 35–60 Venus+ neurons were pooled for each sample, which were immediately lysed in 50 μl of extraction buffer (PicoPure RNA isolation kit, Arcturus, Life Technologies) and total mRNA was subsequently isolated. cDNA was synthesized using Ovation RNA-Seq System V2 kit (Nugen). We obtained approximately 6 μg of cDNA from 35–60 cells from each group. the cDNA library was prepared using Ovation Ultralow DRMultiplex Systems (Nugen). Sequencing was conducted on an Illumina HiSeq2000.
attributes.tag: GEO Accession
attributes.value: GSM2258080