**2.9. RNA extraction and cDNA preparation**

Following exposure to ketamine or control conditions, total RNA was extracted using an RNeasy mini kit (Qiagen, Valencia, CA). Briefly, the cells were rinsed with PBS and lysed with QIAzol lysis reagent (Qiagen). Chloroform was added and the lysates were centrifuged. The upper phase was removed and RNA was precipitated from it with 100% ethanol. The RNA was collected using the RNeasy spin columns (Qiagen). The samples were rinsed with buffer RPE. To elute the RNA from the column, 30 μL of RNase-free water (Qiagen) was added. An Epoch nanodrop spectrophotometer (BioTek Instruments Inc., Winooski, VT) was used to assess the quantity and quality of the RNA in each sample. Each RNA sample was then diluted to 100 ng/μL in RNase-free water. The RNA was reverse transcribed to cDNA using the miScript II RT kit (Qiagen) following the manufacturer's directions. Briefly, a mixture containing 1 μg of RNA, RNase-free water, 10× miScript nucleics mix, reverse transcriptase mix and 5× HiSpec buffer (Qiagen) was prepared. The RT reaction mixture was incubated for 1h at 37°C and for 5min at 95°C to stop the reaction. The RT product was diluted in 200 μL in RNase-free water which yielded a final RNA concentration of 4.5 ng/μL in each sample.
