Long-Read Sequencing Sample Preparation Guidelines

DNA SAMPLE SUBMISSION GUIDELINES

Cell Lines

Collect cultured walled cells or suspension cells, remove culture medium; wash with PBS 1-2 times quickly, remove PBS by low-speed centrifugation; transfer cells into enzyme-free 1.5 ml EP tubes, centrifuge to remove supernatant and place in liquid nitrogen or -80 °C refrigerator for snap-freezing and transport on dry ice.

Note: ≥5×10⁷ cells are required per library. The DNA yield of different cell types varies. In general, the DNA yield of larger cells is slightly less, such as myofibroblasts and neurofibroblasts, and the cell volume can be increased appropriately.

Blood

Fresh whole blood samples are collected directly using EDTA anticoagulated blood collection tubes, mixed 10 times with gentle inversions, and then placed in liquid nitrogen or -80 °C refrigerator and shipped on dry ice after being anticoagulated.

Note:

• Erythrocyte nucleated blood: ≥500 μl whole blood per library.
• Erythrocyte non-nucleated blood: ≥1.5 ml whole blood per library.
• DNA yield varies greatly among species and individuals, with erythrocytes from birds or fish containing nuclei and slightly higher DNA yield, while erythrocytes from human and mammalian blood do not have nuclei and have lower DNA yield, so the sample volume should be increased accordingly.

Animal Tissues

1. Fresh tissues taken from living organisms (or within 10 minutes after the death of the organism) were selected and rinsed with pre-cooled saline or PBS as soon as possible after sampling to remove blood stains and dirt.
2. Connective and adipose tissues and other tissue types not required for the study were quickly removed on ice, and the samples were divided into small pieces of about 30-50 mg (the size of a green bean grain, the smaller the tissue, the better the preservation effect).
3. Blotted dry and placed in enzyme-free in enzyme-free 1.5 /2.0 ml EP tubes or centrifuge tubes.
4. Place in liquid nitrogen or -80°C refrigerator after snap-freezing and transport on dry ice.
5. Note:

• Each library requires ≥1.5 g of animal tissue.
• The DNA yield varies widely among different types of tissues, e.g. DNA yield is higher for metabolically active liver tissues and lower for muscle tissues, which is not recommended.
• Blood or liver samples are recommended for fish and poultry.

Plant Samples

It is recommended to use fresh tissue samples, which should be rinsed with 70% ethanol or sterile water, blotted dry, placed in liquid nitrogen or -80°C refrigerator, then placed in pre-cooled 50 ml centrifuge tubes or wrapped in tin foil and placed in self-sealing bags and shipped on dry ice. More than 2 g per library is required.

Microalgae

Fresh algae are collected, transferred to enzyme-free 1.5 ml EP tubes, washed once with 0.3 M EDTA solution or 70% ethanol, supernatant removed, snap frozen in liquid nitrogen or -80 °C refrigerator, and shipped on dry ice. More than 2 g per library are required.

Microbial Samples

Bacteria, including fungi, actinomycetes and bacteria, should be collected from logarithmic growth stages, removed from the culture medium and other impurities, transferred to 1.5 ml EP tubes without enzymes, snap frozen in liquid nitrogen or -80 °C refrigerator, and transported on dry ice. More than 2 g per library are required.

RNA SAMPLE SUBMISSION GUIDELINES

Cell Lines

Collect suspension or walled cells into enzyme-free EP tubes/centrifuge tubes and remove supernatant by centrifugation at low speed. 1 × 10⁵-10⁷ cells plus 1 ml TRIzol reagent, lyse by repeated blowing to avoid clumping, incubate at room temperature for 5-10 min to fully lyse into homogenate, then freeze at -80 °C and transport on dry ice.

Blood

1. It is recommended that whole blood be isolated and delivered using specialized Blood RNA Tubes, which contain specific reagents for RNA fixation that protect RNA molecules from degradation by RNA enzymes and minimize alteration of in vivo changes in gene expression.

2. Fresh blood is collected using Blood RNA Tubes containing 2.5 ml of blood per tube (a, erythroid nucleated blood: ≥2.5 ml whole blood per library; b, erythroid anucleated blood: ≥5 ml whole blood per library).

3. Gently inverted and mixed 8-10 times immediately after collection.

4. Placed upright at room temperature for 2 - 72 hours and transported on dry ice.

5. After 2 - 72 hours, transport on dry ice.

Animal Tissues

1. Tissues taken in vivo are rinsed with RNase-free saline or PBS to remove blood stains and dirt, and the samples are divided into small pieces of about 30-50 mg, submerged in liquid nitrogen for snap-freezing, placed in enzyme-free EP tubes or centrifuge tubes after thorough freezing, stored in liquid nitrogen or -80°C, and transported on dry ice.

2. If TRIzol lysis solution is used to preserve the sent samples, firstly, liquid nitrogen is ground into powder, then TRIzol is added for lysis, the reference dosage is 50-100 mg/ml TRIzol, after 5 min of lysis at room temperature, -80 °C for freezing and dry ice for transportation.

3. If RNAlater is used, follow the instructions of RNAlater. Keep the tissue block size at 5 mm, too small is not conducive to sample collection, too large the protective solution cannot penetrate the tissue evenly.

Plant Samples

It is recommended to use fresh tissue samples. After sampling, the material should be quickly rinsed with RNase-free DEPC-H₂O or 75% ethanol to remove dust and soil from the surface of the material, blotted dry, and split the sample into small pieces of about 50-100 mg, snap frozen in liquid nitrogen, placed into RNase-free EP tubes or centrifuge tubes, about 500 mg per tube, frozen at -80 °C, and transported on dry ice. More than 3-4 g of fresh plant tissue is required per library.

Microalgae

After collecting fresh algae, transfer to enzyme-free 1.5 ml EP tubes and wash once with RNAase-free 0.3 M EDTA solution or 70% ethanol to remove supernatant. Each library requires ≥3 g of algae.

Microbial Samples

Solid medium fungus, sealed by sealing film and transported at room temperature. For fungi cultured in liquid medium, centrifuge the fungus into centrifuge tubes and immediately flash-freeze in liquid nitrogen, store at -80°C and transport on dry ice. Approximately two 90 mm plate volumes/50 ml of freshly cultured fungal bodies are required per library.