Mammalian Cultured Cells
High molecular weight genomic DNA (HMW gDNA) can be extracted from fresh and frozen cultured cells (adherent or suspension). Fresh cells contain high levels of RNA, but the two-step lysis approach utilized in the cell protocol ensures that RNA is efficiently degraded before the gDNA is released into the lysate and before Proteinase K, which degrades the RNase A enzyme, is introduced. Typically, yields from fresh samples range between 9 and 13 µg for 1 x 106 cells, which is significantly higher (typically ~50%) than yields typically obtained with silica spin columns.
For storage of cell culture samples, it is recommended to prepare aliquots of the desired cell count, pellet by centrifugation, shock freeze, and store at -80°C. Yield and purity of frozen cell samples are comparable to those of fresh cells, but a slight reduction in read length may be observed in ligation-based nanopore sequencing.
Cell samples that are stored in stabilization reagents (e.g., Monarch DNA/RNA Protection Reagent (NEB #T2011), DNA/RNA Shield, RNAlater®) can also be processed, though it is recommended to work with fresh or frozen samples without stabilization reagents for optimal yield, purity and longer, more uniform DNA fragment size.
For samples stored in RNAlater, the cells are still intact and not yet lysed. Pellet the cells, carefully remove the RNAlater, and then follow the standard protocol. For samples stored in 2X Monarch DNA/RNA Protection Reagent or in 2X DNA/RNA Shield, cells are already lysed. Dilute the reagent to 1X with nuclease-free water and use this in place of the Nuclei Prep and Lysis Buffer combination for sample lysis. Following the 10-minute incubation with Proteinase K, add RNase A and incubate the sample at 56°C for 5 minutes. Subsequently, follow the standard protocol.
Blood collected in the presence of common anticoagulants (EDTA, citrate and heparin) can be processed, and both fresh and frozen blood samples can be used. Blood preps are initiated with an erythrocyte lysis step, which helps obtain high yields, maximal purity, and high quality HMW DNA. However, during this process, extended exposure of leukocytes to the RBC Lysis Buffer results in a significant reduction of viability. Therefore, it is important to carry out the RBC lysis steps as quickly as possible and avoid any wait times between steps. Once the leukocytes are washed with PBS, their condition is stable.
Storage and Stabilization of Blood Samples
Regardless of the anticoagulant used, blood samples are processed effectively and without any notable differences in the results. When heparin is used, leukocyte pellets may be more difficult to resuspend, but yield and purity are unaffected. PAXgene® DNA Blood stabilization tubes provide excellent results for both fresh and frozen blood samples; leukocytes are efficiently stabilized in these tubes, and slightly higher overall yields have been observed when compared with other anticoagulants.
Fresh Blood Samples
It is preferable to work with fresh blood whenever possible. Blood sample quality decreases with each day of storage, and leukocyte stability, DNA yield, and length of the isolated DNA fragments will decline as a result. Additionally, as leukocyte stability declines, an increase in “stickiness” of the cells may be observed, requiring more effort to resuspend the pellet completely. Blood samples older than a week should not be used. Before processing, fresh blood samples should always be inverted several times or vortexed briefly to ensure an even suspension of the cell population.
Frozen Blood Samples
If blood samples need to be frozen for later processing, samples should be aliquoted immediately after collection in appropriately sized volumes and containers and should be snap frozen in liquid nitrogen. Frozen aliquots should accommodate the addition of 3 volumes of RBC Lysis Buffer; working in aliquots of 500 µl is recommended for most sample types and allows samples to be processed in 2 ml tubes.
During the freezing process, ice crystals damage the leukocyte cell walls, and upon thawing, cellular DNA becomes vulnerable to nuclease activity. Snap freezing ensures maximal viability of the leukocytes and will lead to higher yields, maximal DNA integrity and better results (read lengths) in ligation-based nanopore sequencing. Frozen samples collected in blood tubes with common anticoagulants (EDTA, citrate, heparin) should, therefore, always be thawed in the presence of cold RBC Lysis Buffer, which prevents damage to the gDNA. One exception is when working with samples frozen in PAXgene DNA Blood Tubes, where leukocytes are efficiently protected from nucleases. These samples do not require thawing in the presence of RBC Lysis Buffer; thawing can be performed according to the manufacturer’s guidance (37°C for 15 minutes), and samples can subsequently be aliquoted and mixed with cold RBC Lysis Buffer as indicated in the protocol.
When processing frozen samples, it is important to use cold RBC Lysis Buffer and to move swiftly through the thawing process to limit the exposure of the cells to warm temperatures. It is also essential to resuspend any cell clumps that remain at the end of the thawing process by vortexing; cell clumps that are not resuspended will result in partially insoluble DNA at the end of the prep. Resuspension of leukocyte pellets from frozen samples is more challenging than with fresh samples, as previously frozen leukocytes have a stronger tendency to clump.
Leukocytes frozen from freshly collected blood samples are robust and typically survive the freeze and thawing process quantitatively. However, leukocytes of aging blood samples exhibit decreased stability, which will result in reduced yields and reduced DNA fragment length. In some cases, using frozen samples instead of fresh samples may result in up to a 35% reduction in yield; in other cases, the difference may be minimal.
Human blood samples can vary significantly among donors in terms of their erythrocyte and leukocyte content, as well as their leukocyte stability. Typically, DNA yields obtained from human samples range from 2.5 µg/100 µl for samples with low leukocyte counts to 6.5 µg/100 µl for leukocyte-rich samples.
Working with animal blood is usually comparable to working with human blood. However, some animals, like horse, rabbit, and guinea pig have extremely high hemoglobin content, while others, like cow, have lower hemoglobin content; this is often noticeable in the dilute appearance of cow blood samples. Leukocyte content can also differ significantly, resulting in variability in DNA yields among different species. For example, pig and rhesus monkey samples typically yield high amounts of DNA (~8 µg and 10 µg per 100 µl, respectively), while cow and mouse samples typically yield lower amounts (1–2 µg per 100 µl).
Rabbit blood is extremely rich in erythrocytes, posing a challenge for complete removal of hemoglobin and other proteins. For optimal purity, a reduced input of 200 µl is recommended. Erythrocyte lysis is not effective for frozen rat samples. Consequently, for rat blood, only fresh samples should be used.
Nucleated Blood (birds, reptiles, fish)
Erythrocytes from non-mammalian vertebrates (birds, reptiles, fish) contain nuclei, and therefore have significantly higher DNA content. As such, input amounts should be reduced by 2 orders of magnitude compared to mammalian blood. Both fresh and frozen nucleated blood can be processed.
- Monarch HMW DNA Extraction Kit for Cells & Blood Manual
- Choosing Input Amounts for the Monarch HMW DNA Extraction Kits
- Choosing an Agitation Speed During Lysis with the Monarch HMW DNA Extraction Kits
- Homogenization of High Molecular Weight DNA (HMW DNA) Samples
- Measuring, Analyzing & Storing High Molecular Weight DNA (HMW DNA) Samples
- Considerations and Performance Data for Nanopore Sequencing of High Molecular Weight DNA (HMW DNA)
- Troubleshooting Guide for High Molecular Weight DNA Extraction Using the Monarch HMW DNA Extraction Kits