title: GSM4471659: Mouse-Cortex_HyPBase_scRNA; Mus musculus; OTHER
library_construction_protocol: Mouse cortical tissues were dissociated to single suspensions following a modification of previously published methods (Avey et al., 2018; Saxena et al., 2012). We incubated samples in a papain solution containing Hibernate-A with 5% v/v trehalose, 1x B-27 Supplement, 0.7 mM EDTA, 70 µM 2-mercaptoethanol, and 2.8 mg/ml papain. After incubation at 37ºC, cells were treated with DNaseI, triturated through increasingly narrow fire-polished pipettes, and passed through a 40-micron filter prewetted with resuspension solution: Hibernate-A containing 5% v/v trehalose, 0.5% Ovomucoid Trypsin Inhibitor, 0.5% Bovine Serum Albumin (BSA), 33 µg/ml DNaseI (Worthington), and 1x B-27 Supplement. The filter was washed with 6 ml of resuspension solution. The resulting suspension was centrifuged for 4 minutes at 250 g. The supernatant was discarded. The pellet was then resuspended in 2 ml of resuspension solution and resuspended by gentle pipetting. We eliminated subcellular debris using gradient centrifugation. We first prepared a working solution of 30% w/v OptiPrep Density Gradient Medium mixed with an equal volume of 1x Hank's Balanced Salt Solution (HBSS) with 0.5% BSA. We then prepared solutions of densities 1.057, 1.043, 1.036, and 1.029 g/ml using by combining the working solution with resuspension solution at ratios of 0.33:0.67, 0.23:0.77, 0.18:0.82, and 0.13:0.87, respectively. We layered 1 ml aliquots of each solution in a 15 ml conical tube beginning with the densest solution on the bottom. The cell suspension was added last to the tube and centrifuged for 20 minutes at 800g at 12ºC. The top layer was then aspirated and purified cells were isolated from the remaining layers. These cells were then resuspended in FACS buffer: 1x HBSS, 2 mM MgCl2, 2 mM MgSO4, 1.25 mM CaCl2, 1 mM D-glucose, 0.02% BSA, and 5% v/v trehalose. Cells were centrifuged for 4 minutes at 250 g, the supernatant was discarded, and the pellet was resuspended in FACS buffer by gentle pipetting. Cells were then sorted based on fluorescence activity. As a gating control, we analyzed cells from cortices injected with AAV9-PB-SRT-tdTomato only. We then collected cells from brains transfected with AAV9-PB-SRT-tdTomato and AAV9-HyPBase whose fluorescence values exceeded the gate. After sorting, cells were centrifuged for 3 minutes at 250 g. The supernatant was discarded and cells were resuspended in FACS buffer at a concentration appropriate for 10x Chromium 3' scRNA-seq library preparation. Single cell RNA-seq libraries were prepared using 10x Genomics' Chromium Single Cell 3' Library and Gel Bead Kit. Each replicate was targeted for recovery of 6,000 cells. Library preparation followed a modified version of the manufacturer's protocol. We prepared the Single Cell Master Mix without RT Primer, replacing it with an equivalent volume of Low TE Buffer. Gel-in-emulsion (GEM) generation and GEM-RT incubation proceeded as instructed. At the end of Post GEM-RT cleanup, we added 36.5 µl Elution Solution I and transferred 36 µl of the eluted sample to a new tube (instead of 35.5 µl and 35 µl, respectively). The eluate was split into two 18 µl aliquots and kept at –20ºC until ready for further processing. One fraction was kept for single cell calling cards library preparation, while the other half was further processed into a single cell RNA-seq library. We added the RT Primer sequence to the products of the scRNA-seq aliquot. We created an RT master mix by adding 20 µl of Maxima 5X RT Buffer, 20 µl of 20% w/v Ficoll PM-400, 10 µl of 10 mM dNTPs, 2.5 µl RNase Inhibitor and 2.5 µl of 100 µM 10x_TSO. To this solution we added 18 µl of the first RT product and 22 µl of ddH2O. Finally, we added 5 µl Maxima H Minus Reverse Transcriptase, mixed by flicking, and centrifuged briefly. This reaction was incubated at 25ºC for 30 minutes followed by 50ºC for 90 minutes and heat inactivated at 85ºC for 5 minutes. The solution was purified using DynaBeads MyOne Silane following 10x Genomics' instructions, beginning at “Post GEM-RT Cleanup – Silane DynaBeads” step D. The remainder of the single cell RNA-seq protocol, including purification, amplification, fragmentation, and final library amplification, followed manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM4471659