GSM6604816: evercode whole-transcriptome library 3; Homo sapiens; RNA-Seq
For the post-mortem samples, each human brain was left to partially thaw and removed from the storage cassette. A small slice of the superior frontal gyrus (approximately 50-100 mg) was cut and transferred to a glass 2 mL dounce tissue homogenizer on ice. For the human biopsies, no structure was visible, and a small slice (approximately 50-100 mg) was transferred to a glass 2 mL dounce tissue homogeniser. All following steps were carried out on the ice and using ice-cold solutions. All centrifugation steps were performed at 500 g for 5 minutes at 4oC using a pre-chilled centrifuge unless otherwise specified. 1.5 mL of Nuclei EZ prep was added, and tissue was homogenised using 20 strokes of the loose and 20 strokes of the tight pestle. The suspension was transferred to a 2 mL tube and 0.5 mL of Nuclei EZ prep was added. The suspension was incubated for 5 minutes and then centrifuged. The supernatant was discarded, 2 mL of Nuclei EZ prep were added, and the pellet was resuspended using a P1000 pipette. The suspension was incubated for 5 minutes and then centrifuged. The supernatant was discarded, 0.5 mL of wash buffer (1x PBS, 1% BSA (15260037; Gibco), 0.2 u/μL SUPERase In (AM2694; Invitrogen)) were added without resuspending, and the sample was incubated for 5 min to allow buffer interchange. Then 1.5 mL of wash buffer was added, and the sample was resuspended. The suspension was centrifuged, the supernatant was discarded, the pellet was resuspended in 500 μL wash buffer, and 0.5 mL of 50% OptiPrep Density Gradient Medium solution (D1556; Sigma-Aldrich) was added. The suspension was transferred on top of a 1 mL 29% OptiPrep cushion solution in a new tube and centrifuged at 10,000g for 30 min at 4oC. The supernatant was discarded, and nuclei were resuspended in 750 μL Parse Nuclei Buffer (from the Parse Evercode WT kit) + 0.75% Bovine Albumin Fraction V (15260037; Gibco). Preparation then proceeded following step 7 of the Parse nuclei fixation protocol (page 15 of the protocol; section 7.5.3).  The following steps were performed using the Parse Evercode nuclei fixation kit (Parse Biosciences) according to the manufacturer's instructions. The Parse Evercode Whole Transcriptome kit (Parse Biosciences) contains all consumables and a detailed protocol that includes all steps from nuclei fixation up to sequencing, including catalogue numbers of reagents. All steps were carried out according to manufacturer's instructions. Suspension preparation and split-pool barcoding were carried out in a BSL-3 laboratory. To move the sample to a BSL 2 laboratory, the following prion decontamination procedure was followed: at the end of the Parse Evercode WT user manual chapter 3.4, the resulting solution was incubated with 3 volumes of TRI-reagent at room temperature for 2 hours (R2050-1-50; Zymo Research). The mix was then transferred out of the BSL-3 facilities and nucleic acids were purified using the Direct-zol DNA/RNA miniprep kit (R2080; Zymo Research). The kit columns were substituted with Zymo-Spin IC Columns (C1004-50; Zymo Research) so that smaller elution volumes could be used, following the advice of Zymo customer support. 11 μL of the RNA fraction and 10 μL of the DNA fraction were eluted in the same tube. The resulting solution of 21 μL was used for the PCRs starting at section 3.5 of the Parse Evercode WT user manual.