Full Service [Library preparation + Sequencing Run]

A. Sample Preparation Pure DNA and RNA are crucial to ensure consistent library preparation and quality data. As recommended by PacBio library preparation guidelines, DNA samples should generally:

• be double-stranded (single-stranded DNA cannot be used to generate the sequencing template)
• be purified by AMPure beads after standard nucleic acid purification
• have not undergone vortexing or multiple freeze-thaw cycles as they can lead to DNA damage
• have not been exposed to high temperatures (e.g. > 65℃ for 1 hour) or pH extremes (< 6 or > 9)
• have an OD260/OD280 ratio of 1.8 to 2.0 and OD260/OD230 ratio of ~2.0
• have not been exposed to intercalating fluorescent dyes or ultraviolet radiation. If gel purification is required, avoid using ethidium/UV based visualization methods. One alternative is to use SYBR^® Safe (Invitrogen) and visualize with blue light
• not degraded, i.e. high-molecular weight, with high DIN score on tapestation
• do not contain denaturants (e.g., guanidinium salts or phenol) or detergents (e.g. SDS or Triton-X100), RNA contaminants, or other insoluble materials
• do not contain carryover contamination from the original organism/tissue (e.g. heme, humic acid, polyphenols, etc.)
• be dissolved in neutral, buffered solution (e.g. 10 mM Tris Acetate or Tris-HCl, pH 8). Avoid eluting in RNAse-free H₂O or unbuffered solutions
• PCR products should be clean amplicons, without non-specific products or multiple bands

RNA samples should generally:

• be purified by AMPure beads after standard nucleic acid purification
• have not undergone vortexing or multiple freeze-thaw cycles
• have not been exposed to high temperatures (e.g. > 65℃ for 1 hour) or pH extremes (< 6 or > 9)
• have an OD260/OD280 ratio of 1.8 to 2.0 and OD260/OD230 ratio of ~2.0
• not degraded, i.e. high-molecular weight, with high RIN score (>=9.0) on bioanalyzer
• do not contain denaturants (e.g., guanidinium salts or phenol) or detergents (e.g. SDS or Triton-X100), RNA contaminants, or other insoluble materials
• do not contain carryover contamination from the original organism/tissue (e.g. heme, humic acid, polyphenols, etc.)

More recommendations:

• Use column purification
• Treat DNA with RNase
• Treat RNA with DNase

B. Sample Requirements Both DNA and RNA need to be quantified by fluorometric-based methods, e.g. PicoGreen or Qubit. They should be assessed by UV spectrophotometry for purity. Before sample preparation, please contact platform specialists to confirm sample submission logistics. We will keep samples properly and return any leftover. If your samples do not meet above requirements, please contact us and we can seek alternatives together.

C. Sample Submission Please submit service request through iLab to obtain project quotation and confirmed the service content before sample submission. Please contact our colleagues in advance (via phone/email shown on left) before bringing the samples to the Core. With your samples, please bring along the following items:

• Hardcopy of completed PB Sample Submission Form, and the softcopy as attachment in iLab
• For DNA samples, gel photo with appropriate size marker