Cell Tissue Culture
1. Culture GM12878 cells in RPMI media supplemented with 15% non heat-inactivated FBS and 2mM L-Glutamax.
2. Grow cells to a density of 1E6 / ml before subsequent dilution of ⅓ every ~3 days, and expand to 9 x T75 flasks (45 ml of media in each).
3. Centrifuge cells for 10 min at 100 x g (4C), wash in 1/10th volume of PBS (pH 7.4) and combine for homogeneity.
4. Evenly split cells between 8 x 15ml tubes and pellet at 100 x g for 10 mins at 4C. Unless proceeding directly with RNA isolation, snap freeze in liquid nitrogen and immediately store at -80C.
1. Add 4 ml of TRI-Reagent to a frozen pellet of 5E7 GM12878 cells and briefly vortex immediately.
Incubate at room temperature for 5 minutes.
2. Add 400 μl BCP (1-Bromo-3-chloro-propane) or 200 μl CHCl3 (Chloroform) per ml of sample, vortex, incubate at room temperature for 5 minutes, briefly vortex again, and centrifuge for 10 minutes at 12,000 x g (4C).
3. Pool the aqueous phase in a LoBind Eppendorf tube and combine with an equal volume of isopropanol.
4. Mix sample, incubate at room temperature for 15 minutes, and centrifuge for 15 minutes at 12,000 x g (4C).
5. Remove supernatant, wash RNA pellet with 750 μl 80% ethanol and centrifuge for 5 minutes at 12,000 x g (4C).
6. Remove supernatant. Air dry pellet for 10 minutes, resuspend in nuclease free water (100 μl final volume), quantify with Qubit or Nanodrop, and either store at -80C or proceed to poly-A purification.
Poly(A) RNA isolation
1. Dilute 100 μg aliquots of total RNA in 100 μl of nuclease free water and poly-A select using NEXTflex Poly(A) Beads.
2. Elute resulting mRNA in Nuclease free water and store at -80oC.
1. Perform first strand cDNA synthesis using Superscript IV and 100 ng of poly-A purified RNA combined with 0.5 ng of the SIRV set 3 control.
2. Perform reverse transcription and strand-switching with primers provided by ONT in the SQK-PCS108 kit.
3. After reverse transcription, amplify with LongAmp Taq Master Mix under the following conditions: 95C for 30 seconds, 11-15 cycles (95C for 15 seconds, 62C for 15 seconds, 65C for 15 minutes), 65C for 15 minutes, hold at 4C.
4. Perform 15 cycle PCR when using the SQK-PCS108 kit and 11 cycle PCR when using the SQK-LSK308 kit.
5. Purify PCR products using 0.8X AMPure XP beads, elute in nuclease-free water.
MinION native RNA sequencing of poly-A RNA
1. Prepare biological poly-A RNA (500-775 ng) and a synthetic control (Lexogen SIRV Set 3, 5 ng) for nanopore direct RNA sequencing generally following the ONT SQK-RNA001 kit protocol, including the optional reverse transcription step recommended by ONT.
2. Replace Superscript III (ONT recommended) with Superscript IV for reverse transcription.
MinION sequencing of cDNA
1. Prepare cDNA sequencing libraries using 1 μg of cDNA following the standard ONT protocol for SQK-PCS108 (1D sequencing) or SQK-LSK308 (1D^2 sequencing).
2. Use 0.8X AMPure XP beads for cleanup, instead of recommended 0.4X.