| GSM6666158 channel 1 growth |
Mice were anesthetized deeply with isoflurane and euthanized via rapid decapitation. The hippocampus and neocortex were dissected out of the brain on a cold surface using clean surgical tools, placed into cryo-safe nuclease-free microcentrifuge tubes, flash frozen in liquid nitrogen (LN2), and stored at –80C until processing. Frozen tissue was then pulverized in LN2-cooled stainless steel Cryo-Cups using cold stainless steel pestles and transferred into cold nuclease-free microcentrifuge tubes and immediately stored at –80C until use. |
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| GSM6666158 channel 1 extraction |
Mouse brain tissue was isolated, flash frozen, and pulverized as described above. Approximately 20mg of pulverized brain tissue per sample was used to isolate RNA. 1mL of TRIzol (Invitrogen 15596018) was added to each nuclease-free eppendorf tube containing pulverized tissue, and cells were lysed via trituration with a P1000 pipette tip, followed by trituration with a 21G and then a 25G needle on a 1mL syringe. Samples were centrifuged at 4°C for 5min at 10,000rcf to remove tissue debris. The supernatant was removed and added to a new tube containing 200uL of chloroform, which were mixed by inversion and cooled on ice for 5min. Samples were centrifuged at 4°C for 15min at 10,000rcf, and the upper aqueous phase containing RNA was transferred into a new tube, followed by the addition of 100uL of isopropanol to precipitate RNA and overnight incubation at -20°C. The next day, samples were centrifuged at top speed (18,000rcf) for 20min at 4°C to pellet the RNA. The pellets were washed twice with 1mL of ice-cold 70% molecular biology grade ethanol and then air dried for 15min at RT. The RNA pellets were resuspended in 20uL of nuclease-free water. On-column DNAse digestion and RNA clean-up was then performed using the Qiagen RNeasy mini kit (Qiagen, Inc. 74106) per manufacturer’s instructions, followed by elution in nuclease-free water. RNA concentration was assessed using Qubit® RNA BR Assay Kit (Q10210) and a Qubit® 3.0 Fluorometer. RNA integrity was assessed using an Agilent 4150 TapeStation system and associated RNA screen tape reagents (Agilent 5067-5576). Only samples with an estimated RNA integrity number (RIN) ≥7.0 were sent to the New York Genome Center (NYGC) for bulk total RNA sequencing. Upon receipt at NYGC, RNA samples were re-evaluated for quantification and integrity, using Ribogreen and Fragment Analyzer 5300, respectively. Total RNA libraries were prepped using Kapa Total library prep with Ribo-Erase, in accordance with manufacturer recommendations. Briefly, 500ng of total RNA was used for ribosomal depletion and fragmentation of total RNA. Depleted RNA underwent first and second strand cDNA synthesis. cDNA was then adenylated, ligated to Illumina sequencing adapters, and amplified by PCR (using 9 cycles). The cDNA libraries were quantified using Fragment Analyzer 5300 (Advanced Analytical) kit FA-NGS-HS (Agilent, catalog number DNF-474-1000) and Spectramax M2 (Molecular Devices) kit Picogreen (Life Technologies, catalog number P7589). Libraries were sequenced on an Illumina NovaSeq sequencer, using paired end sequencing (2 x 100 bp cycles) to a depth of >75M read pairs per sample. |
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