GSM9468883: Old_1; Mus musculus; OTHER
Ovaries were washed in cold PBS and individually submerged in optimal cutting temperature compoud (OCT). Tissues were moved to a cryo-mold with fresh OCT and oriented for sagittal tissue sectioning. Tissue blocks were then flash frozen at -70°C. After freezing, tissue blocks were acclimated to -13°C in a Cryostat NX70 (Thermo Fisher Scientific) for 30 minutes prior to sectioning. Tissue blocks were then mounted on a cutting block with OCT and sectioned at a 5° cutting angle with 10-μm section thickness. Regions of interest (multiple oocytes on the same plain) were identified prior to tissue capture. Four histological cryo-sections sections from 2 female mice for each age cohort were individually placed on the slide-seq puck slides. Pucks with adhered tissue were then removed from the cryostat and placed into a 1.5-ml Eppendorf tube containing 200 µL hybridization buffer (6X SSC, 2 unit/µL Lucigen RNase inhibitor) for 30 minutes at room temperature to facilitate the binding of mRNA to the spatially barcoded beads of the puck. First strand cDNA synthesis was performed in RT solution (115 µL water, 40 µL 5X Maxima RT buffer, 20 µL 10 mM dNTPs, 5 µL RNase Inhibitor, 10 µL 50 uM Template Switch Oligo, 10 µL Maxima H-RTase) for 30 minutes at room temperature, followed by 1.5 hours at 52°C. After reverse transcription, the tissue was removed by adding 200 µL 2X Tissue Digestion Buffer (100 mM Tris pH 8.0, 200 mM NaCl, 2% SDS, 5 mM EDTA, 32 unit/µL Proteinase K) and incubating at 37°C for 30 minutes. Following this incubation, 200 µL wash buffer (10 mM Tris pH 8.0, 1mM EDTA, 0.01% Tween-20) was then added and the mixture pipetted to remove the beads from the coverslip. A series of 3 washes was performed on the beads using 200 µL wash buffer for the first two washes and 200 µL 10mM Tris-HCl (pH 8.0) for the last wash, pelleting for 3 minutes at 3000 RCF, removing the supernatant, and resuspending beads. Samples were then treated with exonuclease I solution (170 µL water, 20 µL ExoI buffer, 10 µL ExoI) for 50 minutes at 37°C. A series of 2 washes was performed on the beads using 200 µL wash buffer and pelleting for 3 minutes at 3000 RCF, removing the supernatant and resuspending. Samples were then resuspended in 0.1N NaOH and incubated for 5 minutes at room temperature followed by a series of 2 washes in 200 µL wash buffer. The beads were resuspended in 200 µL TE, pelleted, the supernatant removed, resuspended in Second Strand Mix (133 µL water, 40 µL 5X Maxima RT buffer, 20 µL 10 mM dNTPs, 2 µL 1mM dN-SMRT oligo, 5 µL Klenow Enzyme) and incubated for 1 hour at 37°C. The beads were washed 3 times with 200 µL wash buffer, resuspended with 200 µL water, and then pelleted for 3 minutes at 3000 RCF. The final resuspension of the tissue digestion was in 200 uL of 10mM Tris-HCl (pH 8.0). The final resuspension prior to Second Strand Synthesis was in TE.  The 200 uL cDNA PCR master mix consisted of  88µL H2O, 100uL Terra PCR Direct Buffer, 4uL Terra Polymerase (Takara, 639270), 4 µL 100 uM Truseq PCR handle primer, and 4 µL 100 µM SMART PCR primer. The final cDNA purification was eluted in 20µL of H2O. Subsequent library preparation was performed using the Nextera XT kit (Illumina, FC-131-1096), starting with 600pg of cDNA using a specific P5-Truseq PCR hybrid oligo in place of the Nextera XT i5 adapter, according to manufacturer's instructions. The PCR product was purified twice with 0.6X AMPure XP beads (Beckman Coulter, A63881), resuspended in 20 µL water, and checked for quality and quantity using the Bioanalyzer (Agilent) and Qubit Fluorometer (Thermo Fisher Scientific). Libraries were pooled and sequenced as 42-bp pair-end reads on four Illumina NextSeq 500 flow cells on the NextSeq 500 instrument using NextSeq Control Software 2.2.0.4 with the following paired read length: 42 bp Read1, 8 bp i7 Index and 42 bp Read2. Following sequencing, Illumina Primary Analysis version NextSeq RTA 2.4.11 and cellranger mkfastq (4.0.0) were run to demultiplex reads for all libraries and generate FASTQ files. Alignment, filtering, barcode, and unique molecular identifier (UMI) counts were done using cellranger count (4.0.0). The barcode-gene matrices were filtered with >=10 unique molecular identifier (UMI) counts using the unique barcode list. Each puck received approximately 400,000 to 500,000 spatial beads, corresponding to 175 genes per bead.