GSM8758789: AAV9_Null_Plasma_BiolRep2; Mus musculus; OTHER
For RNA isolation, cultured NPCs and EVs from conditioned media were processed using the miRNeasy Micro Kit (Qiagen, #217084, Germantown, MD, USA). For the isolation of exRNA from conditioned media, as little as 500 mL from a confluent 48-well plate to 50 mLs from multiple T75 flasks can be used. Conditioned media should on the cells for 48 hours before isolation. Nanodrop was employed for RNA sample quantification, followed by reverse transcription using the Superscript IV VILO gDNA extraction from cultured hNPCs utilized the remaining sample post-removal of the aqueous layer during RNA isolation. The protocol, adapted with modifications from the TRIzolTM Reagent. Experimental protocol for DNA isolation involved the addition of 300 μL of 100% Isopropanol to the sample. After a 30-min incubation at RT, the sample underwent centrifugation for 5 min at 2000 x g at 4°C to pellet the DNA. The DNA pellet was resuspended in 1 mL of 0.1M Sodium Citrate in 10% ethanol and incubated for 30 min and then spun at 2000 x g for 5 min at 4°C. This step was repeated once. The pellet was then resuspended in 1 mL of 75% ethanol and incubated for 10-20 min. Following a 5-min centrifugation at 2000 x g at 4°C, the pellet was air-dried and resuspended in 8 mM NaOH. pH adjustments were made as needed with HEPES. Isolated gDNA and cDNA were amplified for NGS analysis using appropriate primers. A portion of the PCR product was run on a gel to check for band size and appropriate amplification. PCR product was cleaned using the QIAquick PCR purification kit (Qiagen, #28106) and eluted in 35 μL of RNAase free water. Samples were sent for Next Generation sequencing (MGH CCIB DNA Core). After sequencing, the results were analyzed using CRISPResso2.