title: GSM8122742: BALI_multiomic_DNA_DG_vs_CA1_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. All steps were performed under RNAse-free conditions. 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 for 3 times for 5 minutes. The sections were heated for 5 minutes in pre-warmed PBS at 65C in a hybridization oven, and then snap-cooled in ice-cold PBS on ice for 10 minutes. For each slide, an first RT mix (14uL) was prepared containing 2.5 uL of dNTPs (stock 10 mM each), 1.5uL of phosphorilated RT primer (BL730, 100 uM stock) in water, denatured for 5 minutes in 65C and the snap cooled on ice for >2 minutes. A second RT mix (36 uL) was prepared with 10 uL of 5x SuperScript-IV buffer, 2.5uL SuperScript-IV enzyme, 1 uL of RNAsin, 3uL of 100 uM TSO oligo (BL617),  5 uL of 5M Betaine, 2.5 uL of 100 mM DTT in nuclease free water, and added to the first mix, up to a total RT reaction volume of 50uL. Any excess PBS was removed from the slides, and 50 uL of RT mix was added directly on top of the sections, which were then covered with a 22x22 mm coverslip and incubated overnight (~16hrs) at 42C in a sealed humid chamber. The slides were retrieved from the RT reaction, washed one in PBS then twice in 2xSSC for 5 minutes each.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, CA1), BL732/BL641 (cycle 1, single ligation bulk). The annealing thermocycler conditions were as follows: 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 (CA1), 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 (200 uL) was collected and an aliquote (150 uL) was treated with 10 uL of 100 mM PMSF for 10 minutes at room temperature to inactivate the proteinase K prior to biotin pulldown to separate the Tn5 fragments (biotinylated) from cDNA. For each sample, 10uL of myOne C1 beads were washes 3 times in 1xB&W-T buffer, and then resuspended in 150 uL of 2xB&W-T buffer. The lysate was then added and incubated with the beads for 60 minutes at room temperature. After this incubation we collected the supernatant as our cDNA enriched fraction for RNA libraries, while the beads represented our Tn5 fragments enriched fraction for ATAC libraries. The beads were washed 3 times in 1xB&W-T buffer for 5 minutes, once in 1xST buffer for 5 minutes and then directly used as input for library amplification via precise PCR as follows. For each sample, a 50 uL PCR mix was prepared mixing 25 uL of 2x Q5 mastermix, 2.5 uL of 10 uM P5 primer (BL727), 2.5 uL of 10 uM indexed P7 primer (Ad2.X) in nuclease free water, and adding it directly to the washed bead pellet. 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 (after removal of the beads), 5 uL of 2x Q5 master mix, 0.5 uL of 10 uM P5 primer (BL727), 0.5 uL of 10 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 (1.2x) 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.  Separately, the cDNA fraction was purified using Monarch Reaction clean-up columns and eluted in 12.5uL. The cDNA was treated with RNAseH by adding 1.5 uL of 10x RNAseH buffer and 1 uL RNAseH enzyme to each sample, and then incubating the mix at 37C for 30 minutes, 65C for 20 minutes. The cDNA was amplified to via precise PCR as follows. For each sample, a 50 uL PCR mix was prepared mixing 15 uL of RNAseH treated sample, 25 uL of 2x Q5 mastermix, 2.5 uL of 10 uM forward primer (BL753), 2.5 uL of 10 uM reverse primer in nuclease free water. The reaction was incubated in a thermocycler at 98C for 3 minutes, then 5 cycles at 98C for 15 seconds, 67C for 20 seconds, 72C for 60 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, 0.5 uL of 10 uM forward primer (BL753), 0.5 uL of 10 uM reverse primer (BL752), 2.5 uL of 20x EVA-Green in nuclease free water. The cycling protocol was 98C for 3 minutes, 40 cycles of 98C for 15 seconds, 67C for 20 seconds, 72C for 60 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. A small aliquote (2 uL) was analysed on a Tapestation. The amplified cDNA fraction was then tagmented with Tn5 loaded with a single adapter (BL371/BL515) in a reaction set up as follows: 50 ng of pre-amplified cDNA, 0.5uL loaded Tn5 enzyme in 10 mM Tris-HCl pH 7.6, 5 mM MgCl2, 10% Dimethyl Formamide, 0.33x PBS, 0.1% Tween-20, 0.01% Digitonin in 50uL final. The tagmentation reaction was incubated at 37C for 30 minutes, and inactivated adding 50 uL of 80 mM EDTA and 2.5uL of 2% SDS solution for 15 minutes at room temperature. The reactions were purified were purified with AmpureXP beads (0.8x) and eluted in 20 uL of EB buffer. A small aliquote (2 uL) was analysed on a Tapestation. The purified reaction was amplified to generate ATAC libraries as follows: 18 uL of Tn5-cDNA, 2.5 uL of 10uM P5 primer (BL727), 2.5 uL of 10 uM indexed P7 primer (Ad2.X) in nuclease free water. The cycling protocol was 72C for 5 minutes (gap-fill), then 98C for 1 minutes and 5 cycles of 98C for 10 seconds, 68C for 20 seconds, 72C for 45 seconds, with a final extension at 72C for 5 minutes. 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.