Name: GSM5934730
Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: For RNA extraction, cultures and slices were held separately in a chamber on a motorized stage on a motorized microscope Olympus BX61WI, Gibraltar Burleigh Thorlabs. Cultures were superfused 2-4 ml/min in a bath solution of in mM: 145 N-methylglucamine NMG, 2 CaCl2, 1 MgCl2, 10 HEPES, 60 glucose pH 7.3, osmolality 300, in 95% O2-5% CO2 . All procedures were performed with a Multiclamp 700B patch-clamp amplifier Molecular Devices, LLC and using borosilicate glass pipettes with filament, OD 1.5 mm, ID 1.10 mm, Sutter and the input resistance was in the range of 3-9 MΩ. RNA was extracted with a 1440A Digidata AD board Molecular Devices, LLC under pClamp v10. Electrodes pipettes were filled with in mM: 100 KCl, 35 N-methylglucamine NMG, 10 EGTA, 1 CaCl2, 4 MgCl2, and 10 HEPES pH 7.3, 290 mOsm for the RNA extraction from the TOV-21G cells. Electrodes were filled with in mM: 130 K-gluconate, 10 KCl, 10 HEPES, 10 EGTA, 2 MgCl2, 2 Na-ATP, 0.2 Na-GTP, and 5 glutathione pH 7.3, 280-290 mOsm for the RNA extraction from the Thy1-YFP L5 neurons. For the RNA extraction from the Thy1-GCaMP6f L2/3 neurons, electrodes were filled with in mM: 110 K-gluconate, 4 KCl, 4 NaCl, 0.2 CaCl2, 10 HEPES, 1.1 EGTA, 2 Mg-ATP, 1 MgCl2, and 5 glutathione pH 7.2-7.3, 300 mOsm. Membrane voltages were corrected for an estimated liquid junction potential of about 10 mV. Perforation was performed using -escin antibiotics 50 M from a stock solution 25 mM respective for each cellular population. All solutions were made with nuclease-free, de-ionized, not DEPC-treated water Ambion AM 9938. Lucifer yellow 50 mM was added to the electrode intracellular working solution for all cellular populations. The intercellular working solution including the antibiotics and dye was prepared fresh daily and used at room temperature and were backfilled into the electrodes for the patch-clamp experiments. Holding potentials for TOV-21G cells and cortical neurons were -80 mV and -70 mV, respectively. Electrodes were made by a PP-830 Narishige puller and positive pressure was applied as the pipettes were advanced to the cell of interest. The leak current Ilk was then used for the holding current in the current-clamp protocol and it was noted before and after the current was injected in all cellular populations. Seven current injection steps of 5 pA each was applied for a duration of 500 ms per step for the RNA extraction. Following RNA extraction, electrodes were slowly retracted while leaving the cell intact in its native environment, and the pipette solution was collected for sequencing. Each sample tube was prefilled with 2 µL of IGEPAL working mixture per tube, and positive pressure was applied to eject the internal contents of the electrode into a sample tube. The IGEPAL working mixture contained 65 µL nuclease-free de-ionized water Ambion, AM9938, 25 µL RNaseOUT™ Recombinant Ribonuclease Inhibitor ThermoFisher, 10 µL IGEPAL CA-630 Alfa Aesar, J61055 and was stored in 4°C for up two weeks. RNA was collected in 0.5 mL tubes Protein LoBind Tube 0.5 mL, Eppendorf, 022431064. After RNA extraction, samples were transferred on dry ice and stored at -80°C. After each RNA extraction, the electrode holder and silver-chloride recording electrode were cleaned with 70% ethanol. Cytosolic extraction for all cellular populations was performed by first doing whole-cell patch-clamp, then applying negative pressure light suction to harvest cellular content into IGEPAL working mixture. The cDNA libraries were prepared using Ovation SoLo RNA-Seq System Tecan Genomics. UMIs are used to eliminate possible PCR duplicates in sequencing datasets and facilitate unbiased gene expression profiling. For some experiments, ERCC RNA Spike-In Mix Thermo Fisher Scientific was included in the library preparation. The processed libraries were assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit Agilent Technologies. The libraries were pooled and loaded onto a TruSeq SBS v3 flow cells on an Illumina HiSeq 2500 Illumina or an S1 flow cell on an Illumina NovaSeq 6000 Illumina , and run for 2X50 cycles according to the manufacturer's instructions. De-multiplexed and adapter-trimmed sequencing reads were generated using Illumina bcl2fastq version 2.18.0.12 allowing no mismatches in the index read.