GSM6604824: 40dpi split-seq library 2; Mus musculus; RNA-Seq
Flash-frozen mouse brain was left to partially thaw. The olfactory bulb removed, and a small slice of the frontal cortex was cut and transferred to a glass 2 mL dounce tissue homogenizer on ice. All following steps were carried out on 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. 2 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 15 mL tube and 2 mL of Nuclei EZ prep was added. The suspension was incubated for 5 minutes and then centrifuged. The supernatant was discarded, 4 mL of Nuclei EZ prep was added, and the pellet was resuspended using a P1000 pipette. The suspension was incubated for 5 minutes and then centrifuged. The supernatant was discarded, and the pellet was resuspended in 4 mL Nuclei Suspension Buffer (NSB; 1x PBS, 0.01% BSA (Cambridge Bioscience; 227-10210) and 0.1% NxGen RNAse inhibitor (Lucigen; 30281-2)). The suspension was centrifuged, the supernatant discarded, and the pellet resuspended in 1 mL NSB. The suspension was filtered through a 35 um filter (Fisher Scientific; 10585801), and stored on ice. For nuclei fixation, 3 mL of 1.33% formalin solution were added to the 1 mL of nuclei suspension. The suspension was incubated on ice for 10 minutes. 169 uL of 5% Triton-X was added to the fixed nuclei, the solution was mixed by pipetting and then incubated on ice for 3 minutes. Nuclei were centrifuged at 500g for 5 minutes at 4oC, supernatant was discarded, and the pellet was resuspended in 500 uL cold NSB. 500 uL of cold 100 mM Tris and 20 uL of 5% Triton-X were added. Nuclei were centrifuged again under the same conditions; supernatant was discarded, and the pellet was resuspended in 400 uL of cold 0.5X PBS. Nuclei were filtered through a 35 um filter, counted using a Neubauer-Improved haemocytometer, and diluted to a final concentration of 2000 nuclei/uL using cold 0.5x PBS supplemented with 0.2 units/uL SUPERase In RNAse inhibitor. The nuclei were stored frozen at -80oC until library preparation. Sequencing libraries from the mouse brain samples were prepared using the SPLiT-seq protocol (version 3) (Rosenberg et al. 2018). A summary of the methodology and optimisations is given below. For the reverse transcription, 4 uL the first 24 wells of Stock plate 1 were transferred to a new PCR plate on ice. 8 uL of RT mix (per reaction: 4 uL Maxima 5x RT buffer, 0.124 uL NxGen RNAse inhibitor, 0.25 uL SUPERase In, 1 uL 10 mM Takara dNTPs, 2 uL Maxima H minus enzyme, 0.625 uL H2O) was added to each well and then 8 uL of fixed nuclei suspension. The plate was placed in a thermocycler and PCR was carried out as per protocol instructions. All wells were then pooled together, Triton-X was added to a final concentration of 0.1% and suspension was centrifuged for 3 minutes at 500g. The supernatant was discarded, and nuclei were resuspended in 2 mL 1x NEBuffer 3.1 (New England Biolabs; B7203S) + 20uL NxGen RNase Inhibitor. For ligation round 1, the ligation mix (1337.5 uL water, 500 uL 10x T4 ligase buffer (included with ligase enzyme), 100 uL T4 DNA Ligase (New England Biolabs; M0202L), 100 uL BSA 10 mg/mL, 12.5 SUPERase In, 40 uL NxGen RNAse inhibitor) was added to the nuclei suspension and into a basin. 40 uL of the suspension were pipetted in each well of the Ligation round 1 barcode plate and the plate was incubated in a plate shaker at 37oC for 30 minutes and 300 rpm rotation. Then 10 uL of the Ligation Round 1 blocking solution (316.8 uL 100 uM BC_0216, 300 uL 10x Ligase buffer, 583.2 uL water) were added to each well and the plate was incubated again under the same conditions. For ligation round 2, the nuclei suspensions were pooled in a 15 mL Falcon and passed through a 40 um strainer to another Falcon. 100 uL T4 DNA ligase was added and the mix was transferred to a basin. 50 uL of the mix was added to each well of the Ligation Round 2 barcode plate. The plate was incubated as previously. Then 20 uL of the Ligation Round 2 blocking solution (369 uL 100 uM BC_0066, 800 uL 0.5 M EDTA, 2031 uL water) was added to each well. The wells were pooled in a 15 mL Falcon and passed through a 40 um strainer into another Falcon. 70 uL 10% Triton-X was added to the mix and nuclei were centrifuged for 5 minutes at 1000g. The supernatant was aspirated, and nuclei were washed with 4 mL wash buffer (4 mL 1x PBS, 40 uL 10% Triton-X, 10 uL SUPERase In) and centrifuged again under the same conditions. Supernatant was aspirated and nuclei were resuspended in 100 uL 1x PBS + 2 uL SUPERase In. Nuclei were counted using a Neubauer-Improved haemocytometer and a desired number of them was aliquoted in each 1.5 mL Eppendorf tube. PBS was used to fill each tube up to 50 uL. Each tube is referred to as a “sublibrary”. For nuclei lysis, 50 uL 2x lysis buffer (final concentrations: 20 mM Tris pH 8, 400 mM NaCl, 100 mM EDTA pH 8, 4.4% SDS (Thermo Fisher Scientific; AM9822)) were added to each tube followed by 10 uL 20 mg/mL Proteinase K. The mix was incubated at 55oC for 2 hours with shaking at 300 rpm. Lysates were frozen at -80oC and processed the following day. 5 uL 100 uM AEBSF (Abcam; ab141403) was used to stop proteinase reaction. Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific; 65002) were washed and used to bind the barcoded transcripts as per protocol. Beads were resuspended in 200 uL Template Switch mix (88 uL water, 44 uL 5x Maxima buffer, 44 uL 20% Ficoll PM-400, 22 uL 10 mM Takara dNTPs, 5.5 uL NxGen RNAse inhibitor, 5.5 uL 100 μM Template Switch Oligo, 11 uL Maxima H minus enzyme) and incubated at a rotating incubator at room temperature for 30 minutes and at 42oC for 1.5 hours. The sample was then washed, resuspended in PCR mix (121 uL 2x Kapa Hifi Master Mix, 9.68 uL 10 uM BC_0108, 9.68 uL 10 uM BC_0062, water up to 242 uL) and split equally in 4 different wells of a PCR plate. The following PCR programme was then used: 3 min at 95oC, then 20 s at 98oC, 45 s at 65oC, 3 min at 72oC for a total of 5 cycles, and then hold at 4oC. Reactions were combined in a single tube, cleaned with 0.6x AMPure XP beads as per manufacturer's instructions, eluted in 20 uL water, mixed with 180 uL of the same PCR mix and split in 4 wells (50 uL per well). 2.5 uL EvaGreen (Biotium; #31000) was added to each well and amplification continued in a QuantStudio 7 Flex qPCR machine (Thermo Fisher Scientific) until signal plateaued out of exponential amplification using the following programme: 3 min at 95oC, then 20 s at 98oC, 20 s at 67oC, 3 min at 72oC until signal plateaus out of exponential amplification, then 5 min at 72oC, hold at 4oC. Reactions were combined in a single tube, cleaned with 0.6x AMPure XP beads as per manufacturer's instructions, eluted in 10 uL water and analysed at TapeStation 2200 using a gDNA tape. 600 pg of each sample was used for tagmentation using the Nextera XT sample prep kit using custom primers one of BC_0076-BC_0083 and BC_0118, according to manufacturer's instructions. Resulting libraries were analysed on a TapeStation 2200 using a high sensitivity D1000 tape or a high sensitivity D5000 tape.