GSM6734946: multiplexed mitochondria library: L5R29P1B; Mus musculus; OTHER
A single neuron or astrocyte was harvested using a micropipette from a coverslip of the Mitotracker labeled neuron cells under the microscope (40x magnification, Olympus IX70), and then was placed in the 1.7 mL Eppendorf Tube with 4 μl of lysis buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 2% CHAPS, PH 8, and freshly added Proteinase inhibitor cocotail). After spinning down briefly, the Eppendorf Tube was incubated on ice for 10 minutes. Then 15 μl of mitochondrial buffer (220 mM sucrose, 68 mM mannitol, 10 mM KCl, 5 mM KH2PO4, 2 mM MgCl2, 0.5 mM EGTA, PH 7.2; 0.01% BSA and 1X Proteinase inhibitors cocktail) was added into the Eppendorf Tube. The isolated mitochondria from single neuron or astrocyte were now ready for the flow cytometry sorting preparation. Single mitochondria were sorted in each of the wells of a 96-well plate containing 4 uL of lysis buffer (1x TE, 0.1M NaCl, 2mM CaCl2). 1 uL of 0.25 ug/uL Proteinase K was added to each well and incubated at 55 °C for 30 mins. The Proteinase K inactivation was performed by incubation at 95 °C for 10 mins. The plates were rested on ice until the next step. Nuclease free water and 2.5 uL reaction buffer (10X) was added to each well followed by incubation at 70 °C for 3 mins. The plates were immediately rested on ice for at least 2 mins to complete denaturation. After addition of the random primers (40 ng/uL), the plate was incubated at 25 °C for 50 minutes. Bovine serum albumin (0.1 mg/mL), dNTPs (2mM), DTT (10mM) and Phi 29 DNA (5U) polymerase was added to each well. The 25 uL reaction mixture was incubated at 30 °C for 28 hours. Another 5U of Phi 29 DNA polymerase was added to the reaction mixture at 20 hours to continue the reaction. The reaction was stopped after 32 hours by incubation at 65 °C for 10 mins. The RCA product was stored at -20 °C until the next step. Each single mitochondrion was then amplified with 12 different SNVs using the same Non-Seq Barcode Mx (x=1-10) in front of its genomic forward primer. The amplification reaction on each single mitochondrion was set up as follows in a 25 μl Volume:  1μl of mtDNA amplified by Rolling Circle Amplification (RCA) from single cell, 1μl of 24 oligos pool for 12 SNVs amplification (1.25μM), 23 μl Platinum PCR SuperMix (Invitrogen, 12532-016).  The amplification was performed on a T100 thermal cycler (BioRad) using the following program: 94℃ 2 min for the 1st cycle, followed by 25 cycles of 94℃ 30s, 49℃ 30 s, 68℃ 30 s, and then added another 15 μl Platinum PCR SuperMix for another 20 cycles using the same PCR program as the previous 25 cycles, and finally hold at 4℃. After the amplification, each single mitochondrion was analyzed on the Agilent Type Station 4150 using an Agilent High Sense DNA kit. Then the 10 single mitochondria (M1 through M10) were pooled together and cleaned up by 1.5X AMpure XP beads (A63881, Beckman Coulter). The 50 ng of the pool was input into a library construction using TruSeq Nano DNA kit (Illumina, 20015964) per the manufacturer's instructions. Libraries were combined into a sequencing pool and then run on Illumina NextSeq 500 with 150bp single-end reads.