Materials, Instrument and Methods
Materials: The eCAP ssDNA 100-R kit (PN 477480, Figure 1B) and LIF Performance Test Mix (PN: 726022) were from SCIEX, Framingham, MA. The Human HPRT Single Guide RNA (sgRNA) (100 nt, PN 217541712) was obtained from IDT, Coralville, Iowa. CleanCap Cas9 mRNA with modification (PN L7206-20, 20 µg) was from TriLink BioTechnologies, San Diego, California. Urea (PN 29700), Nuclease free water (PN AM9932) and SYBR Green II RNA gel stain, 10,000x concentrate in DMSO (PN S7564) were obtained from Thermo Fisher Scientific, Waltham, MA. Polyvinyl-pyrrolidone (PVP, PN 437190), Transcript RNA markers 0.2-10kb (PN R7020) and 10xTBE buffer (PN 574795), Molecular Biology Grade were from SigmaAldrich, St. Louis, MO. The 0.2 µm syringe filter (PN 4602) was from PALL Corporation, Port Washington New York. Rainin LTS filter tips were from Mettler Toledo, Oakland, California.
Instrument and software: A PA 800 Plus Pharmaceutical Analysis System (Figure 1A) equipped with LIF detector and solid-state laser with excitation wavelength at 488 nm and a 520 nm band pass emission filter were from SCIEX, Framingham, MA. Data acquisition and analysis were performed using 32 Karat software V10.
Tris-Borate-7 M Urea Buffer Preparation: This step must be done one or a few days before running samples. To rehydrate the buffer, 135 ml of 0.2 µm filtered deionized water was added to the bottle containing dry tris-borate in the ssDNA 100-R kit. The solution was mixed with a clean stirring bar for about 30 min or until boric acid was completely dissolved. Then, the entire amount of urea from the urea bottle in the ssDNA 100-R kit was added while the solution was being stirred. The urea was completely dissolved after about 2 hours and the solution became clear. This buffer should be good for one month if stored at 2oC to 8oC. Before sample run, the required amount of buffer was removed and filtered through a 0.2 µm filter. SYBR Green II dye was added at a 1 to 50,000 dilution. About 3.5 ml of this dyecontaining buffer was used for each set of 6 injections.
Preparation of 100-R gel: Five milliliters of freshly filtered TrisBorate- 7 M Urea buffer was added to the lyophilized gel. The solution was stirred for about 3 to 6 hours until the gel is completely dissolved. Alternatively, the gel solution was placed on a LABQUAKE rotator (Barnstead International, Dubuque, IOWA) in a cold room (2oC to 8oC) for 72 hours with gentle rotation. For analysis of single guide RNA (100 nt) and pd(A) 40- 60 Test Mix, undiluted 100-R gel was used as is. For analysis of cas9 mRNA and large RNA molecules, 536 µl of the 100-R gel was diluted 2.8 fold to 1500 µl with filtered Tris-Borate-7 M Urea buffer. After thorough mixing by gentle pipetting and swirling, the diluted gel was spun on a Qualitron DW-41 micro centrifuge at 2000 x g (6400 rpm) for 4 min to remove bubbles.
Cartridge Assembly: DNA capillary (PN 477477) was installed per instructions on kit insert (PN 726479) in the ssDNA 100-R kit. The total capillary length was 40.2 cm with 30 cm as the effective length.
LIF Calibration: To ensure consistent response of LIF detector throughout this study, the LIF detector was calibrated using LIF Calibration Wizard and Performance Test Mix (PN: 726022). following the instructions in LIFluor EnhanCE user’s guide (PN 725824). This calibration was done whenever capillary or LIF detector or the laser was changed.
Preparation of Buffer Trays and Sample Trays: Vial positions for buffer trays are indicated in Figure 3 with rows 1 to 3 for tests with guide RNA and pd(A) 40-60 Test Mix and rows 4 to 6 for tests with Cas9 mRNA and larger RNA molecules. All solutions were pipetted with filter tips. Each “NF Water” vials were filled with 1.5 ml nuclease free (NF) water. Waste vial was filled with 1 ml NF water. “Dilut. 100R” vial was filled with 1.5 ml diluted 100- R gel. “100R Gel” vial was set up with a microvial containing 210 µl 100-R gel. “Sep Buffer” vials were filled with 1.6 ml filtered Tris-Borate-7 M Urea buffer containing SYBR Green II dye.
Sample Preparation: Filter tips were used for all steps. Lyophilized sgRNA was resuspended in 10 mM Tris, pH 7.5, 0.1 mM EDTA to a concentration of 100 µM or 3.246 µg/µl and stored at -80oC in 10 µl aliquots. Before use, it was then diluted with nuclease free water to 25 ng/µl and treated at 80oC for 2 minutes. Immediately after heat treatment, it was placed on ice for 5 minutes before being transferred to a microvial (PN 144709) and loaded onto the sample inlet tray. The pd(A) 40-60 Test Mix from the 100-R kit was resuspended in 500 µl of nuclease free water and stored at -20oC in 50 µl aliquots. It was further diluted 2 fold with NF water before injection. The Sigma Transcript RNA markers and Cas9 mRNA were aliquoted upon first thawing and stored at -80oC in aliquots. Before use, they were diluted to 25 ng/µl (marker) and 10 ng/µl (Cas9 mRNA), and treated at 65oC for 5 minutes, followed by 5 minutes on ice before transferred to a microvial and loaded onto the instrument.
Instrument Set up: Conditions used for analysis of sgRNA and pd(A) 40-60 Test Mix were as described in Figures 4-8. Separation was done at 30 kv for 50 minutes for sgRNA and at 12.4 kv for 50 minutes for the pd(A) 40-60 Test Mix. Conditions for analysis of Cas9 mRNA and the Transcript RNA markers were carried out as shown in Figures 4 to 6 and 8-9.