title: GSM8122747: BALI_RNA_SVZ_vs_CTX_e165; Mus musculus; OTHER
library_construction_protocol: For embryo collection, pregnant dams were euthanised by CO2 overdose with exposure to gas in rising concentration followed by cervical dislocation. Embryos were euthanised by cooling on ice followed by decapitation. Heads were frozen in OCT resin by immersion in a dry ice/isopentane bath followed by storage at -80C. 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). All steps were performed under RNAse-free conditions. Cryosections of embryonic E16.5 brain (coronal plane) 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 (0.5% TritonX100 in PBS) for 15 minutes at room temp, and then washed in PBS 3 times for 5 minutes. The sections were incubated with 0.1N HCl for 5 minutes and washed 3 times in PBS (two quick washes, one 5 minutes wash). The section were heated for 5 minutes in pre-warmed PBS at 65C in an hybridization oven, and then snap-cooled in ice-cold PBS on ice for 10 minutes. From this point onwards all steps performed in light protected conditions. For each slide, an first RT mix (14uL) was prepared containing 2.5 uL of dNTPs (stock 10 mM each), 1.5uL of caged RT primer (BL621, 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, 0.5 uL ET-SSB, 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 index oligos for the ligations were annealed at 45uM final in 2xSCC in the following pairs: BL599/BL623 (cycle 1, SZV) and BL601/BL624 (cycle 1, cortex). 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 slides were retrieved from the RT reaction, washed one in PBS then twice in 2xSSC for 5 minutes each. Nuclei were stained by incubating the section with Draq 5 (1:1000 dilution in 2xSSC) for 10 minutes, and then washed 2 times for 5 minutes in 2xSSC. Since the RT primer was directly caged, we proceed with two consecutive rounds of uncaging/ligation to mark the SVZ and cortex areas (bilaterally). For the uncaging, the slides were mounted with 200 uL of 2xSSC and a thin glass coverslip (22x22mm). The uncaging was performed on a SP5 Leica confocal system fitted with a 30mW 405nm solid state laser, illuminating each ROI for 5 minutes with the 405nm laser at maximum power. The slides were washed in 2xSSC, once quickly and once for 5 minutes, and then 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, alternating BL599/BL623and BL624/BL601 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 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, a 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 light protected anymore. The lysate was collected and purified with MinElute Reaction Clean-up column, and eluted in 25 uL of EB buffer. A small aliquote of the lysate (2uL) was used as input for a qPCR to measure the optimal cycling conditions for the amplification of the libraries. The qPCR reaction was set up as follows: 2 uL of purified lysate, 25 uL of 2x Q5 master mix, 0.25 uL of 50 uM universal forward Truseq primer, 0.5 uL of 50 uM index A012 Truseq primer 2.5 uL of 20x EVA-Green. The cycling protocol was 98C for 3 minutes, 40 cycles of 98C for 15 seconds, 68C for 20 seconds, 72C for 20 seconds, plate reading step. The number of cycles required to reach 1/3 of the maximum signal was selected for the final amplification of the libraries. The final amplification reaction was set as follows: 10 uL of purified lysate, 25 uL of 2x Q5 master mix, 0.25 uL of 50 uM universal forward Truseq primer, 0.5 uL of 50 uM index A012 Truseq primer, 2.5 uL of 20x EVA-Green. The cycling conditions were the same as the qPCR. 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.