GSM6337547: 1502, 4mg MALAT1 ASO IT; Macaca fascicularis; RNA-Seq
Tissues were collected two weeks after the final dose to allow sufficient time for target knockdown. Brain tissue was dissected and frozen for analysis. Nuclei were isolated from frozen mouse brain tissue samples in a procedure similar to a previously published “Frankenstein” protocol (https://www.protocols.io/view/frankenstein-protocol-for-nuclei-isolation-from-f-bq23mygn). Briefly, frozen tissue samples were placed into chilled Nuclei EZ Lysis Buffer (Sigma Aldrich, # NUC101) and mechanically homogenized using a 2 mL dounce homogenizer by stroking 15-20 times with pestle A followed by 15-20 strokes of pestle B. The lysate was incubated on ice for 5-10 mins, passed through a 70 µm strainer mesh, pellet and resuspended again in Nuclei EZ Lysis Buffer for another 5 mins of incubation on ice. After 3-4 washes to remove debris (500g at 4°C for 5 minutes), nuclei were resuspended in Nuclei Wash Buffer with a final concentration of 0.2 U/uL RNase inhibitor (Ambion, cat. no. AM2682) and 1% BSA in PBS, and then passed through a 30µm strainer.  Using DAPI stain, roughly 25,000 - 35,000 nuclei were FACS sorted on BD FACSAria (Becton Dickinson) directly into 10x Genomics RT master mix for a target recovery of 10,000-15,000 nuclei. RT enzyme was added immediately prior to microfluidic loading on Chromium for nuclei capture, and libraries were prepared per 10X Genomics user guide (CG000204 Rev B). Next-generation sequencing was performed by SeqMatic (Fremont, CA) on an Illumina NovaSeq instrument with a S2 flow cell generating paired-end reads (28x10x10x90 bases).