title: GSM8122640: BALI_DNA_DG_vs_CTX_adult_rep2; Mus musculus; OTHER
library_construction_protocol: For adult brain collection, mice were euthanised by cervical dislocation followed by confirmation of death by permanent cessation of circulation. Animals were then decapitated and the brain dissected and frozen in OCT resin and stored as above. Cryosections were cut using a XXXX cryostat under RNAse free conditions. Cryosections were cut using a XXXX cryostat under RNAse free conditions. The instrument was wiped with RNAse-ZAP prior to use and between different blocks. A different blade was used for each block. Sample blocks were equilibrated to the chamber temperature for 15 minutes prior to sectioning, and then mounted on the object holder. Sections were cut at a thickness of 10 uM and collected on RNAse-free SuperFrost Plus microscope slides (one section per slide). Slides were left at room temperature for 20-30 seconds to help tissue adhesion and then stored on dry ice (short term) or at -80C (long term). The adapters for transposome assembling were annealed at 50 uM each in 1x Annealing buffer (40 mM TrisHCl pH 8.0, 50 mM NaCl) with the following thermocycling protocol: 95C for 5 minutes, slow ramp to 65C (0.1C/sec), 65C for 5 minutes, slow ramp to 10C (0.1C/sec). The oligos pairs used were BL699/BL538 (BALI-adapter, universal ligation root) and BL515/BL538 (A-adapter, single modality ATAC) or BLXXX/BL538 (A-adapter, multi-omics). The transposomes were assembled by mixing 3 uL of BALI-adapter, 3 uL of A-adapter, 6 uL of unloaded Tn5 and incubating the mix at 23C for 30 minutes and then used immediately afterwards. Cryosections of adult brain were retrieved from cold storage and equilibrated for 5 minutes at room temperature and then heated for 5 minutes at 37C on a metal heat block. Pap-pen was used to draw a hydrophobic boundary around each section, and let dry for 5 minutes at room temperature. Sections were incubated with 500 uL PBS to dissolve and remove the OCT embedding matrix, and fixed with 500 uL of 0.5% PFA in PBS for exactly 5 minutes at room temperature. Fixation was quenched by removing the PFA and promptly adding 1.25M Glycine in PBS, one quick wash and one wash for 5 minutes at room temperature. After washing twice in PBS for 5 minutes each, the sections were permeabilised in Permeabilization buffer (10 mM TrisHCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.01% Tween-20, 0.01% IGEPAL, 0.001% Digitonin, 1% BSA, 10 uL murine RNAse Inhibitor) for 15 minutes at room temp. The sections were washed in 1x Wash Buffer (10 mM TrisHCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 10 uL murine RNAse Inhibitor) once quickly and once for 5 minutes, then in PBS twice for 5 minutes. For each section, 200 uL of transposition mix were prepared as follows: 2uL of loaded Tn5 in 10 mM Tris-HCl pH 7.6, 5 mM MgCl2, 10% Dimethyl Formamide, 0.33x PBS, 0.1% Tween-20, 0.01% Digitonin. Each section was sealed with an adhesive hybridization chamber and incubated with 200 uL of tagmentation mix for 35 minutes at 37C in a humid chamber. The slides were retrieved and washed with 40 mM EDTA once quickly, and once for 5 minutes, then with PBS 3 times for 5 minutes and with 2xSSC 2 times for 5 minutes. The index oligos for the ligations were annealed at 45uM final in 1xSSC in the following pairs: BL680/BL676(cycle 1, bulk), BL681/BL677 (cycle 2, bulk), BL683/BL642 (cycle 3, bulk), BL732/BL601 (cycle 4A, Dentate Gyrus), BL733/BL677 (cycle 4B, cortex), BL732/BL641 (cycle 1, single ligation bulk). The annealing thermocycler conditions were as follow: 1) 95C for 5 minutes, 2) ramp down to 65 at 0.1C/second, 3) hold at 65C for 5 minutes, 4) ramp down to 12C at 0.1C/second, 5) hold at 12C.. The spatial barcodes were encoded in the tissue with the following protocol, which was conducted under light protected condition for its entirety: cycle 1 ligation, cycle 2 uncaging (bulk), cycle 2 ligation, cycle 3 uncaging (bulk), cycle 3 ligation, cycle 4A uncaging (Dentate Gyrus), cycle 4A ligation, cycle 4B uncaging (cortex), cycle 4B ligation. Bulk uncaging was performed with a UV transilluminator fitted with 365 nm bulbs for 15 minutes at room temperature, with the section covered with 500 uL of 2x SSC. Spatial uncaging was performed on a SP5 Leica confocal system fitted with a 30mW 405nm solid state laser, illuminating each ROI for 10 minutes with the 405nm laser at maximum power. Following uncaging, the slides were washed in 2xSSC, once quickly and once for 5 minutes. Ligations were performed as follows. Sections were incubated for 5 minutes in 1x Ligation buffer (50 mM TrisHCl pH7.4, 10 mM MgCl2, 10 mM DTT, 1mM ATP). For each sample, a ligation mix (50 uL) was prepared as follows: index oligos (3 uM, as appropriate), T4 ligase (100U/uL, NEB) in 1x T4 ligation buffer. The ligation mix was added directly to the sections, covered with a 22x22mm glass coverslip, and the reactions were incubated for 30 minutes (45 minutes for cycle 1) at 25C in a humid chamber. The slides were then washed in 2xSSC+Draq5 (1:1000) once for 10 minutes, 2xSSC+10%EC once for 5 minutes and 2xSSC once for 5 minutes. After the serial ligations, an adhesive hybridization chamber was mounted on the slides, and the sections were lysed in 200 uL of Lysis Buffer (0.8 U/mL in RIPA buffer) at 60C overnight (~16 hrs) in a sealed humid chamber. All the following steps were not carried out under light protected conditions. The lysate was collected and purified with MinElute Reaction Clean-up column, and eluted in 25 uL of EB buffer. Libraries were amplified via quantitative PCR as follows. For each sample, a 50 uL PCR mix was prepared mixing 5 uL of purified sample, 25 uL of 2x Q5 mastermix, 1.25 uL of 50 uM P5 primer (BL727), 1.25 uL of 50 uM indexed P7 primer (Ad2.X) in nuclease free water. The reaction was incubated in a thermocycler at 72C for 5 minutes (gap-fill), 98C for 3 minutes, then 5 cycles at 98C for 20 seconds, 68C for 20 seconds, 72C for 20 seconds. The pre-amplified products was kept on ice, and 5 uL were used as input for qPCR to determine the optimal number of additional cycles, The qPCR reactions were set up as follows: 5 uL of the preamplified material, 5 uL of 2x Q5 master mix, 0.25 uL of 50 uM P5 primer (BL727), 1.25 uL of 50 uM indexed P7 primer (Ad2.X), 2.5 uL of 20x EVA-Green in nuclease free water. The cycling protocol was 98C for 3 minutes, 40 cycles of 98C for 20 seconds, 68C for 20 seconds, 72C for 20 seconds with a plate reading step. The number of cycles required to reach 1/3 of the amplification curve plateau was selected for the final amplification of the libraries. The PCR reactions were purified with AmpureXP beads (0.8x) and eluted in 20 uL of EB buffer. The libraries were analysed on a Tapestation (D5000 cartridge) and quantified with KAPA library quantification kit prior to pooling. Sequencing was performed on a Novaseq (Illumina) instrument.