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Banana Slug Genome Project A. Dissection 1. Received frozen slug, thawed in 37°C water bath 2. Made careful incision on side of slug, being careful not to break intestinal 3. Isolated and identified various body parts 4. Took out pieces of all major organs/body parts and put them into 5. Stored at -80°C B. Extraction (using Omega E.Z.N.A. Mollusk DNA kit) 1. Took pieces of the digestive gland, albumen gland and kidney and 2. Placed a marble mortar and pestle into a bed of dry ice 3. Poured liquid nitrogen into mortar and placed sample into it, letting it 4. Ground sample to fine powder as the liquid nitrogen boiled off 5. Since the mortar is already cold, sample will remain in powder form even 6. Scooped powdered sample into tube containing buffer ML1 and tissue separate tubes weighed them (25mg kidney, 120mg albumen, 60mg digestive gland) freeze completely 7. Added chloroform:isoamyl alcohol (24:1), centrifuged at 10,000 x g for 2 8. We carefully pipetted the aqueous phase into a clean 1.5mL when all of the liquid nitrogen boils off Proteinase K, and incubated at 60°C until the sample was fully solubilized (Note: albumen and digestive gland were split into two tubes/columns due to mass of sample) minutes. This step removes the mucopolysaccharides from our sample, and leaves an upper aqueous phase containing our soluble DNA. microcentrifuge tube and added buffer MBL and RNase A, and incubated at 70°C for 10 minutes. 9. Once the sample cooled to room temperature we added absolute ethanol 10. Transferred 750ul of each sample to the HiBind® DNA Mini Columns 11. Spun columns at 10,000 x g for 1 minute, discarded filtrate 12. Repeated steps 10 and 11 until all of sample was applied to columns 13. Added 500ul HBC buffer with isopropanol, spun at 10,000 x g for 30sec, 14. Added 700ul DNA Wash Buffer with ethanol, spun at 10,000 x g for 1 15. Repeated steps 13 and 14 once 16. Dry spun for 2 minutes at maximum speed (14,000 x g) 17. Transferred columns to clean 1.5ul microcentrifuge tubes 18. Added 100uL Elution Buffer (preheated to 70°C), let sit at room 19. Stored DNA at 4°C discarded filtrate minute, discarded filtrate temperature for 2 minutes, spun at 10,000 x g for 1 minute C. Illumina Library Prep (Two library preps side by side in two different size ranges) 1. Sheared DNA to two different sizes, ~400bp and ~600bp (6ug into each). 2. Size selected for two ranges, 350-500bp and 500-700bp. 3. Blunt End Repair with 50ul of sample plus the following: * 7.12ul ddH2O * 7ul Buffer Tango (10x) * 0.28ul dNTPs (25mM each) * 0.7ul ATP (100mM) * 3.5ul PNK (10U/ul) * 1.4ul T4 DNA Polymerase (5U/ul) Incubated at 25°C for 15 minutes then at 12°C for 5 minutes Cleaned up reaction with 1.8x 18% PEG SPRI beads Washed 2x with 80% EtOH Eluted DNA in 20ul TE Size selected, blunt ended DNA amounts: 350-500bp: 62.6ng/ul x 20ul = 1,252ng 500-700bp: 20.0ng/ul x 20ul = 400ng 4. Adapter Ligation with 20ul sample plus the following: * 10ul ddH2O * 4ul T4 Ligase Buffer + 10mM ATP (10x) * 4ul PEG-4000 (50%) * 1ul P5+P7 adapters (100uM each) * 1ul T4 DNA Ligase (5U/ul) Incubated at 22°C for 30 minutes Cleaned up reaction with 1.8x 18% PEG SPRI beads (same as last time) 5. Adapter fill-in with 20ul sample plus the following: * 14.1ul ddH2O * 4ul ThermoPol Buffer (10x) * 0.4ul dNTPs (25uM each) * 1.5ul Bst DNA Polymerase (8U/ul) Incubated at 37°C for 20 minutes Cleaned up reaction with 1.5x 18% PEG SPRI beads Washed 2x with 80% EtOH Eluted DNA in 30ul TE Measure concentration by Qubit 300-550bp: 28ng/ul x 30ul = 840ng 550-750bp: 11.8ng/ul x 30ul = 354ng 6. Indexing PCR (index 89 for 300-550bp, index 90 for 550-750bp) Performed 10 cycles of PCR with 200ng of each sample (2 rxns each with 100ng each) Thermal Cycler Parameters * 25ul KAPA 2x Polymerase mix * 2.5ul sample * 1ul Primer IS4 (10uM) * 1ul indexing primer (10uM) * 20.5ul ddH2O 1. 98°C for 3 minutes 2. 98°C for 20 seconds 3. 65°C for 30 seconds 4. 72°C for 40 seconds 5. Return to step 2 for 11 more times 6. 72°C for 3 minutes 7. 4°C forever Measured by Qubit 350-500bp: 53.8ng/ul x 20ul = 1076ng 500-700bp: 60.4ng/ul x 20ul = 1208ng 7. Size selection of PCR product Used E-gel SizeSelect 2% gel, selected for DNA in corresponding size ranges. Measured by Qubit 350-500bp: 4.44ng/ul x 20ul = 88.8ng 500-700bp: 7.36ng/ul x 20ul = 147.2 D. Lucigen Nxseq Long Mate Pair Library Prep Followed instructions exactly, with the exception of manually size selecting on 0.75% Agarose field inversion gel instead of selecting using AMPure beads