TITLE: A cost-effective way of extracting high molecular weight (HMW) DNA from low amounts of fresh or herbarium lichen material 
AUTHORS: Eva Andrea Strasser

[DESCRIPTION]
Presented method allows the extraction of high molecular weight (HMW) DNA of all lichen symbionts using low input (5 - 100 mg) lichen material. Extractions using fresh and over 50-year-old herbarium material have been successful, yielding HMW DNA fragments from 50 to 200 kbp in length suitable for both short- and long-read sequencing. The protocol includes an initial acetone cleaning step to dissolve secondary lichen metabolites, which can later be identified using thin-layer chromatography (TLC). Apart from an overnight incubation during the enzymatic digestion of fungal, algal, and bacterial cells, this protocol provides a relatively fast and easy extraction method. A final size selection is achieved using Solid Phase Reversible Immobilization (SPRI) magnetic beads and a 20%-PEG-0.75M-NaCl buffer.

[BEFORE_START]
Prepare and store enzymes according to the manufacturer's instructions. Thaw enzymes just before use
If Buffer ATL contains precipitates, dissolve by heating to 70 °C with gentle agitation
Prepare liquid stocks (m[g] = Mr * c[mol/L] * V[L] ) and fill up with nuclease-free water to desired volume
For long-read sequencing (fragments > 1 kbp) prepare PEG-NaCl-Buffer: 
        0.75 M NaCl 
        20% PEG: V% = (v/v)% = volume of solute / volume of solution * 100

[GUIDELINES]
Handle samples very gently. Avoid vortexing, instead gently flick the tubes. Use wide-pored pipettes, which can be made by carefully cutting off the tip of a pipette with a sterile razor blade in a clean bench. During extraction, the number of tubes will double. In the last step, they will be combined again. 
For long-read sequencing of HMW DNA use fresh lichen material. To test HMW DNA quantity and quality, capillary electrophoresis is recommended. For short-read sequencing, a quantification using a fluorometer (and gel electrophoresis) is recommended. 
DNA purity can be examined using a UV-VIS spectrophotometer - an absorbance ratio of A260/A280 in the range of 1.8 – 2.0 and A260/A230 higher than 2.0 indicates good quality and purity of the DNA.

[STEPS]
SECTION: ENZYMATIC DIGESTION - DAY 1
3. Add 1 mL Buffer 1 to each sample 
Mix thoroughly by flipping the tube
Incubate for 15 minutes at RT
Centrifuge at 3000 x g for 2 minutes. If necessary repeat at a higher speed (e.g. for crust lichens) until a precipitate forms
Discard supernatant
Add 500 µL Buffer 2 to each sample 
Mix enzymes well before taking an aliquot. Then add 5 µL each of Glucuronidase, Cellulase, Protoplast F, and Lysozyme to each sample
Add 90 µL Chitinase to each sample
Place the tubes in a thermomixer overnight at 35 °C and rotate at   for 9 seconds every 5 minutes
SECTION: ENZYMATIC DIGESTION - DAY 2
4. Set thermomixer to 56 °C
Centrifuge samples with 3000 x g for 2 minutes
Discard supernatant while working under the fume hood
Add 600 µL Buffer ATL and mix slowly and carefully
Add 20 µL Proteinase K and mix slowly and carefully
Incubate in a thermomixer at 56 °C with shaking at   for at least 1 hour
Remove samples and set the thermomixer to 70 °C 
Briefly centrifuge tubes to remove drops from inside the lid
Add 600 µL Buffer AL, close the lid, and mix gently using the lab rocker at  , or by light shaking. Sample and Buffer AL have to be thoroughly mixed to yield a homogeneous solution
For low-input samples, an optional step is to dissolve 1 µg of carrier RNA in 1 µL of buffer ATL. Then, add 1 µL of the dissolved carrier RNA to each sample
Incubate tubes at 70 °C in a thermomixer with shaking at 600 rpm for 10 minutes
Briefly centrifuge tubes to remove drops from inside the lid
Add 300 µL ethanol (96-100%), close the lid and mix by light shaking or on the lab rocker at 5 rpm for at least 10 seconds. Mix samples and ethanol thoroughly to ensure efficient binding
Briefly centrifuge tubes to remove drops from inside the lid
Transfer 700 µL of the supernatant into a labelled 1.5 mL DNA LowBind tube. Transfer the remaining ~800 µL into a second labelled tube, resulting in two storage vessels per sample
SECTION: EXTRACTION AND SIZE SELECTION
5. Thoroughly shake the SPRIselect bottle to resuspend the SPRI beads
Add *1.2 SPRIselect to the sample (sample volume [µL] * 1.2 = SPRIselect [µL]) 
For long-read sequencing, an optional step is to add *0.65 of PEG-NaCl-Buffer (sample volume [µL] * 0.65 = PEG-NaCl-Buffer [µL])
Mix samples gently using the lab rocker, or rock the storage vessels from side to side and incubate at RT for 30 seconds. Mix well, as insufficient mixing will lead to inconsistent results.
Repeat the mixing and incubating step
Place tubes into the magnetic stand and allow the SPRI beads to settle to the magnets (settle times will vary)
Remove the clear supernatant. Be aware that significant bead loss will result in reduced yield
Add 700 µL 5 M NaCl without removing the tubes from the magnetic stand, incubate for 1 minute, and discard the supernatant 
Repeat the previous washing step
Remove the tubes from the magnetic stand and add ≥ 20-70 µL of nuclease-free water (or standard buffer like Tris or TE)
Carefully resuspend the beads by slowly mixing them on the lab rocker or by pipetting ten times using wide-pore pipettes until homogeneous
Incubate at RT for 1 minute
Place the tubes on a magnetic stand, allowing the SPRI beads to settle to the magnets. If the elution volume is too low so that beads cannot settle to the magnet, flip the magnetic stand while holding the reaction vessels in place
Transfer the eluate (HMW DNA) into a labelled 1.5 ml DNA LowBind storage vessel, and combine divided samples
Check DNA quantity and fragment size. If DNA fragments are too short, repeat the size selection part using *0.8 SPRIselect and *1.0 PEG-NaCl-Buffer
SECTION: For long reads (&gt; 1kb) only: PEG-NACl-buffer preparation
8. Prepare 20%-PEG-0.75-M-NaCl-Buffer:
NaCl 58,44 g/mol:  m = M * c * V 
PEG 20%:  V% = (v/v)% = volume of solute/volume of solution * 100 
20 % = 20 mL/100 mL = x mL / 200 mL * 100 
x = 20 mL * 200 mL / 100 mL = 40 mL PEG in 200 mL  
Shake vigorously, vortex and shake again.
SECTION: BUFFER PREPARATION
2. Buffer 1: prepare 1 mL/sample (plus overhang) using liquid stocks
100 mM Tris-HCl
Add 10 mM DTT solution just before use

Buffer 2: prepare 500 µL/sample (plus overhang) under the fume hood using liquid stocks
1 M Sorbitol 
25 mM EDTA 
5.9 mM Citric Acid  
4.1 mM Sodium Citrate 
14 mM ß-Mercaptoethanol
SECTION: SAMPLE PREPARATION
1. If necessary clean lichen material using nuclease-free water and dry the sample
Cut lichen material into small pieces (µm) using a sterile razor blade or scalpel
Transfer samples into 2.0 ml Eppendorf Protein LoBind tubes and store them at -80 °C
Add 400 µL cold acetone (stored at -20 °C), shake gently and remove the supernatant. Supernatant can be stored for use in thin-layer chromatography (TLC)
Repeat until supernatant is clear
Put the tubes in a heating block set at 30 °C under the fume hood until the samples are dry