NextSeq 500 sequencing; RNA-seq of PV+ interneurons in WT and Cox6a2-/- mice to unravel the molecular signaling that contributes to the increase in oxidative stress and metabolic dysregulation following Cox6a2 knockout.
RNA-seq of PV+ interneurons in WT and Cox6a2-/- mice to unravel the molecular signaling that contributes to the increase in oxidative stress and metabolic dysregulation following Cox6a2 knockout.
P15 and P60 WT and Cox6a2-/- mice expressing EGFP under the PV promoter were sacrificed by decapitation and their brains were collected in ice-cold carbogen-bubbled ACSF (120 mM NaCl, 25 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2, and 10 mM Glucose) supplemented with 500 uM Ascorbic acid (brand?). The entire cortex (i.e. from prefrontal to visual cortex) was microdessected on ice and dissociated in 1 mL Papain solution (Papain Dissociation System, Worthington LK003150) containing 500 uM Ascorbic acid, at 37 °C for 30 min. During the incubation, the tissue was mechanically disrupted by gentle pipetting every 10 min to obtain a single-cell suspension. To remove debris and non-dissociated tissue fragments, the cell suspension was filtered through a 70 um cell-strainer and subsequently collected by centrifugation (300 g for 5 min). The collected pellet was resuspended in 1 mL EBSS resuspension buffer (900 uL EBSS buffer, 100 uL ovalbumin inhibitor, 50 L DNase I) and layered over 2 mL ovalbumin inhibitor. Next, the cell suspension was centrifuged at 150 g for 6 min and the obtained cell pellet was resuspended in 300 uL FACS buffer (4.7 mL HBSS buffer, 250 μL BSA (20%), 50 μL DNase I (2 μg/μL)). Single-cell population positive or negative for EGFP signal was independently bulk-collected for RNA extraction on FACS Aria I or III (Becton Dickinson) using a 100 μm nozzle in 350 uL RLT buffer containing 1% beta-mercaptoethanol. Subsequent to collection, samples were vortexed, centrifuged, and stored at -80 °C until further use.  RNA extractions were performed using Qiagen RNeasy kit following the manufacturer's instructions. A total of 250 pg RNA per sample with a RIN value of 7.5-10 was mixed with 0.2 L RNase Inhibitor (40 U/L), 1 L Oligo-dT (10 M, /5Biosg/AAGCAGTGGTATCAACGCAGAGTACT30VN/3/), and 1 L dNTP mix (10 M, 2.5 M each). The sample was centrifuged and incubated at 72 °C for 3 min and processed into cDNA via the Smart-seq2 method as described by Picelli et al. 2014 following some modifications to reverse transcription and cDNA amplification. In short, 5.3 μL reverse transcription master mix containing 0.5 μL SuperScript III (200 U/L), 0.25μL RNase inhibitor (40 U/μL), 2 μL first strand buffer (5x), 2 μL betaine (5 M), 0.06 μL MgCl2 (1 M), and 0.5 μL DTT (100 mM) was added to each sample. The plate was centrifuged for 1 min at 1000 g and incubated for reverse transcription as: 42 °C – 90:00, 42 °C – hold, 42 °C – 12:20, 10 cycles (50 °C – 2:00, 42 °C – 2:00), 39 °C – 10:00, 70 °C – 15:00, 4 °C – hold. After first 90 min of reaction, the plate was removed from the thermocycler and 0.4 μL TSO (12.5 μM, /5Biosg/AAGCAGTGGTATCAACGCAGAGTACATrGrG+G/3/) was added to each sample and incubated for the remaining reaction.  Following reverse transcription, 15 μL cDNA enrichment master mix containing 12.5 μL KAPA HiFi HotStart ReadyMix (2x), 0.25 μL (10 μM, /5Biosg/AAGCAGTGGTATCAACGCAGAGT/3/), and 2.25 μL water was added to each first strand reaction. The plate was centrifuged and incubated according to the following conditions: 98 °C – 3:00, 5 cycles (98 °C – 0:20, 60 °C – 4:00, 72 °C – 6:00), 6 cycles (98 °C – 0:20, 64 °C – 0:30, 72 °C – 6:00), 5 cycles (98 °C – 0:20, 67 °C – 0:15, 72 °C – 7:00), 72 °C – 10:00, 4 °C –hold. Amplified cDNA was purified using AMPure XP beads at a ratio 1:1 as described in (Picelli et al., 2014).   Illumina Nextera XT sequencing libraries were prepared according to the manufacturer's instructions using 350 pg input cDNA per sample and 12 cycles of library enrichment PCR. Concentration of cDNA libraries was determined by Qubit 3.0 according to the manufacturer's instructions and average library fragment length was assessed using the DNA HS assay on a bioanalyser (Agilent). Each library was individually normalized to a final concentration of 2nM and all libraries were combined to yield a 2nM library pool. Equimolar library pools were denatured according to Illumina's recommendations and sequenced on an Illumina NextSeq500 (single end, 75bp) at an average depth of 10mio reads/ library.