GSM8821069: Aged2_PlateSeq; Mus musculus; RNA-Seq
To minimize batch effects, young and aged murine ovaries were collected on the same day for sequencing. Following removal of murine ovaries and receipt of human ovaries, surrounding tissue was removed by fine dissection. Ovaries were gently chopped using a razor blade and then transferred to a Conical tube with 2 mg/mL collagenase type IV (Gibco, 17104019) in DMEM (Gibco, 11960044). Tubes were incubated at +37oC shaking at 250rpm for 20 min then centrifuged at 250 x g for 5min. The supernatant was aspirated and 0.25 % trypsin-EDTA (Gibco, 25200056) was added. Tubes were incubated at +37oC shaking at 250rpm for 20 min then centrifuged at 250 x g for 5 min. The supernatant was aspirated, and the tissue was quenched with 10% FBS (Gibco, 10438026) in DMEM. Using a P1000, tissue was gently triturated to generate a single cell suspension. The cell suspension was filtered using a reversible 37µm sieve (STEMCELL Technologies, 27215). The sieve was rinsed with quench buffer (<37µm filtered flow through) and then flipped and placed in a new tube for rinsing with quench buffer (>37µm unfiltered mature oocyte pool hand-picked for SmartSeq2). Smart-seq2 Library Preparation: Single oocytes were handpicked using an EZ-Grip pipette with a 125 μm EZ-Tip, briefly washed in 0.04% BSA-PBS, and transferred into individual wells containing lysis buffer master mix. Lysed oocytes were snap-frozen on dry ice and stored at −80°C for further processing by the Genomics Platform at the Chan Zuckerberg Biohub SF.   Reverse transcription (RT) and complementary DNA (cDNA) synthesis were performed in 96-well plates using the Smart-seq2 protocol. To preserve RNA integrity, 2–4 lysis plates were thawed on ice and processed in a pre-PCR laboratory. RT master mix (6 μL per well in 96-well plates; 0.6 μL per well in 384-well plates) was prepared using the following components: 0.06 μL 1M MgCl₂, 2 μL 5× First-Strand Buffer (Takara Bio, 639538), 2 μL 5M Betaine, 0.5 μL 100 mM DTT, 0.1 μL 100 μM TSO (Exiqon, 5′-AAGCAGTGGTATCAACGCAGAGTGAATrGrGrG-3′), 0.25 μL RRI, 1 μL SmartScribe Reverse Transcriptase (Takara Bio, 639538), and 0.09 μL water. The RT master mix was dispensed into each well using an SPT Labtech Dragonfly Discovery liquid handler. Plates were sealed with a clear microseal, centrifuged at 1,000 rcf for 1 min, and incubated in a thermal cycler (Applied Biosystems ProFlex or Bio-Rad C1000 Touch Thermal Cycler**) under the following conditions: 42°C for 90 min, followed by 70°C for 5 min.  SmartSeq2 library construction: Second-strand synthesis and cDNA amplification were performed by adding 15 μL of cDNA master mix per 96-well plate (1.5 μL per 384-well plate), containing 12.5 μL KAPA HiFi HotStart ReadyMix 2X (Kapa Biosystems, KK2602), 0.25 μL 10 μM IS PCR primer (IDT, 5′-AAGCAGTGGTATCAACGCAGAGT-3′), 0.113 μL Lambda Exonuclease (NEB, M0262L), and 2.14 μL water. The cDNA master mix was dispensed using an SPT Labtech Dragonfly Discovery, and the plates were sealed, centrifuged at 1,000 rcf for 1 min, and subjected to the following cycling conditions: 37°C for 30 min; 95°C for 3 min; 21 cycles of 98°C for 20 s, 67°C for 15 s, 72°C for 4 min; followed by a final extension at 72°C for 5 min.   cDNA quantity was spot-checked using a Quant-iT dsDNA High Sensitivity Kit (Thermo Fisher, Q33120) and fluorescence was measured using a SpectraMax M5 microplate reader (Molecular Devices). cDNA quality was assessed on an Agilent 5300 Fragment Analyzer. High-concentration cDNA samples were diluted 1:10 with nuclease-free water for optimal enzymatic fragmentation.   Library preparation was performed using Illumina's Nextera XT DNA Library Prep Kit (FC-131-1096), miniaturized for 384-well plates using an SPT Labtech Firefly liquid handler. Briefly, 0.4 μL cDNA (<1 ng) was stamped into each well of a 384-well plate, followed by 1.4 μL of tagmentation master mix (0.2 μL Tagment DNA buffer (TD) and 1.2 μL Amplicon Tagment Mix (ATM)). The plates were sealed, centrifuged at 2,000 rcf for 1 min, and incubated at 55°C for 5 min. Tagmentation was stopped by adding 0.4 μL of Neutralize Tagment Buffer (NT), followed by centrifugation at 2,000 rcf for 5 min.   Index PCR was performed by adding 1.2 μL of Nextera PCR Master Mix (NPM) and 0.8 μL of Nextera index adapters (0.4 μL of 5 μM i5 indexing primer, 0.4 μL of 5 μM i7 indexing primer, IDT). The thermal cycling program consisted of: 72°C for 3 min; 95°C for 30 s; 12 cycles of 95°C for 10 s, 55°C for 30 s, 72°C for 30 s; followed by a final extension at 72°C for 5 min.   Barcoded libraries were pooled using an SPT Labtech Mosquito LV, purified with two consecutive 0.7× AMPure XP bead clean-ups (Beckman Coulter, A63881), and eluted in 30 μL of nuclease-free water. Library fragment size was analyzed on an Agilent 4150 Tapestation System with D5000 ScreenTape (5067-5588) and quantified via qPCR (Bio-Rad CFX96 RT System) using the KAPA Library Quantification Kit (Kapa Biosystems, KK4923).  10X Genomics Library Preparation: Filtered cell suspensions were counted and adjusted to 1,000 cells/μL in 0.04% BSA (Research Products International, A30075) in 1X PBS. Library preparation was performed by the UCSF Genomics CoLabs using the 10X Genomics Chromium Next GEM Single Cell 3' Kit with dual indexing, following the manufacturer's protocol.