TITLE: cf-RRBS protocol
AUTHORS: Andries De Koker, Ruben Van Paemel, Bram De Wilde, Katleen De Preter, Nico Callewaert

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The methylation profile of circulating cell-free DNA (cfDNA) in blood can be exploited to detect and diagnose cancer and other tissue pathologies and is therefore of great diagnostic interest. There is an urgent need for a cost-effective genome-wide methylation profiling method that is simple, robust and automatable and that works on highly fragmented cfDNA. We report on a novel sample preparation method for reduced representation bisulfite sequencing (RRBS), rigorously designed and customized for minute amounts of highly fragmented DNA. Our method works in particular on cfDNA from blood plasma. It is a performant and cost-effective methodology (termed cf-RRBS) which enables clinical cfDNA epigenomics studies.</div></div>

[STEPS]
?. [Lambda spike-in and dephosporylation]
Add to Eppendorf tube:Unmethylated lambda DNA (0.01 ng/µL, 0.1 %w/w): 1 µL or 0.5 µL depending on the DNA input (prepare by going from 10 ng/µL stock to 1 ng/µL to 0.1 ng/µL to 0.01 ng/µL)Recombinant Shrimp Alkaline Phosphatase (rSAP, NEB) (1U/µL): 1 µL10X CutSmart buffer: 1.4 µLDNA: 10.6 µL or 11.1 µLTotal volume: 14 µLPipet up and down to mix DNA with samples. Note: all steps with enzymes should be in a cooling block.
?. [Lambda spike-in and dephosporylation]
Incubate 60 minutes at 37°C.Heat kill the phosphatase for 30 minutes at 75°C.
?. [MspI digest]
Prepare mastermix containing per sample + 1:1 µL mixture containing:  10X CutSmart buffer: 0.1 µLMspI (NEB) (20U/µL): 0.5 µLNuclease-free H20: 0.4 µLAdd 1 µL mastermix to each sample.Total volume: 15 µLPipet up and down to mix DNA with samples.
?. [End-repair and A-tailing]
Prepare mastermix containing per sample + 1:10 µL mixture containing:  Klenow Fragment (NEB, 3' -> 5' exo-) (5U/µL): 0.5 µLdNTP: 0.25 µL  (40µM dATP, 4µM dCTP and 4µM dGTP)10X CutSmart buffer: 1 µLNuclease-free H2O: 8.25 µLAdd 10 µL of the mastermix to each sampleTotal volume: 25 µL
?. [End-repair and A-tailing]
Incubate this 25 µL-mixture:20 minutes at 30°C20 minutes at 37°CHeat kill for 20 minutes at 75°C.
?. [Adapter ligation]
Beforehand, the adapter should be aliquoted and heated to 95°C and afterwards -0.1°C/sec to 4°C. This is to reduce the number of secondary structures. A slow cooling ensures the most stable secondary structure to be formed.Prepare 2 mastermixes containing per sample + 11. Ligase-mixture: 5 µL  T4 DNA ligase (2000U/µL) (NEB): 0.5 µL 10X CutSmart buffer:  0.5 µLNuclease-free H20: 4 µL2. Adapter mixture: 10 µL  Adapter* 10 µM: 1 µLATP (10mM) (NEB): 4 µL10X CutSmart buffer:  1 µLNuclease-free H20: 4 µLKeep the two mixtures separated for as long as possible (or else the T4 ligase can ligate the adapters). Add both mixtures (ligase: 5µL, adapter: 10 µL) to the Eppendorf tube.2. Adapter mixture: 10 µL  Adapter* 10 µM: 1 µLATP (10mM) (NEB): 4 µL10X CutSmart buffer:  1 µLNuclease-free H20: 4 µLKeep the two mixtures separated for as long as possible (or else the T4 ligase can ligate the adapters). Add both mixtures (ligase: 5µL, adapter: 10 µL) to the Eppendorf tube.Keep the two mixtures separated for as long as possible (or else the T4 ligase can ligate the adapters). Total volume: 40 µL*The adapter is identical to the NEBNext-adapter (NEB) (seq: 5´-/Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC/ideoxyU/ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3´) and was synthesized at IDT, but without phosphorotioate bond at the 3’ end. Phosphorothioate bonds are resistant to nuclease degradation, but our cfRRBS protocol is depending on the action of exonucleases (see further).
?. [Adapter ligation]
Ligation was done overnight (14 hours) at 16°C and the ligase was heat killed at 65°C for 10 minutes.
?. [Exonuclease]
Prepare mastermix containing per sample + 1:5 µL mixture containing:  Exonuclease I (20U/µL) (NEB): 0.5 µLExonuclease III (50U/µL) (NEB): 0.5 µLExonuclease VII (10U/µL) (NEB): 0.5 µL10X CutSmart buffer: 0.5 µLNuclease-free H20: 3 µLAdd mixture to Eppendorf tubeTotal volume: 45µLNote: keep enzymes on ice.
?. [Exonuclease]
Incubate for 2 hours at 37°CHeat kill 20 minutes at 95°C.The exonucleases will degrade unligated adapter and partially ligated DNA-fragments but leave completely circular DNA-products derived from MspI/MspI-fragments produced from cfDNA untouched.
?. [Bisulfite conversion]
The MspI/MspI-digested adapter-ligated DNA fragments are bisulfite converted using the EZ DNA Methylation-Lightning™ Kit (ZymoResearch) according to manufacturer’s instructions. More information: https://www.zymoresearch.com/media/amasty/amfile/attach/_D5030T_D5030_D5031_EZ_DNA_Methylation-Lightning_Kit_Instructions_ver_1_0_4_LKN-SW_.pdf
?. [Quantification and sequencing]
Based on the concentration value obtained the libraries were pooled and were sequenced on a NextSeq500 instrument, performing a SE75 run using 15% PhiX with 7 dark cycles if all the samples in the run are of RRBS origin. Else, 1-5% PhiX and no dark cycles are sufficient.
?. [Prepare input DNA]
Label 1 thin-walled Eppendorf (0.2 µL) tube per sample (work in strips of 8).
?. [MspI digest]
Incubate for 30 minutes at 37°C.Meanwhile, aliquot and melt the adapter (step 9).
?. [Prepare input DNA]
Collect DNA samples. The volume should be lower than 10.6 µL*If the standard amount of lambda spike-in is to be added (1 µL of 0.01 ng/µL):Add nuclease-free H2O to a total volume of 10.6 µLIf a lower amount of lambda spike-in is to be added (0.5 µL of 0.01 ng/µL), or if the total DNA is lower than 10 ng:Add nuclease-free H2O to a total volume of 11.1 µLIf a lower amount of lambda spike-in is to be added (0.5 µL of 0.01 ng/µL), or if the total DNA is lower than 10 ng:*If the volume is higher, concentrate the samples using a vacuum centrifuge at 30°C. Don't forget to turn the vacuumpump on.
?. [Endonuclease]
Prepare mastermix containing per sample +1:5 µL mixture containing:  Antarctic Thermolabile UDG (1U/µL) (NEB): 1 µLEndonuclease VIII (10U/µL) (NEB): 0.5 µL10X CutSmart: 0.5 µLNuclease-free H2O: 3 µLAdd mixture to Eppendorf tubeTotal volume: 50 µLUGD cuts a uracyl which results in an abasic site. This abasic site is recognised by endonuclease VIII. This step cuts the hairpin and creates ssDNA (bisulfite requires ssDNA).
?. [Endonuclease]
Incubate for 1 hour at 37°CHeat kill for 10-20 minutes at 75°C.
?. [Final amplification step (KAPA HiFi HotStart Uracil+ ReadyMix PCR Kit (KAPA Biosystems)]
Add mixture to the indices-labelled Eppendorf tubes containing:2X Polymerase mix: 15 µLNEBNext universal primer (IDT): 0.9 µLNEBNext index primer (IDT): 0.9 µLAdd 14 µL of the bisulfite converted sample to the mixture.
?. [Bisulfite conversion]
Add 130 μl of Lightning Conversion Reagent to 25 μl (differs from the manifactors protocol) of a DNA sample in a PCR tube.Mix, then centrifuge briefly to ensure there are no droplets in the cap or sides of the tube.Note: If the volume of DNA is less than 20 μl, compensate with water.
?. [Bisulfite conversion]
Place the PCR tube in a thermal cycler and perform the following steps:98°C for 8 minutes54°C for 60 minutes4°C storage for up to 20 hours. Note:  Replicate reactions can be cleaned using the same column for each by repeating steps 3-5. The capacity of the collection tube with the column inserted is 800 μl. Empty the collection tube whenever necessary to prevent contamination of the column contents by the flowthrough.
?. [Bisulfite conversion]
Add 600 μl of M-Binding Buffer to a Zymo-SpinTM IC Column and place the column into a provided Collection Tube.Note: make sure ethanol was added to the M-Wash buffer (step 29).
?. [Bisulfite conversion]
Load the sample (from Step 2) into the Zymo-SpinTM IC Column containing the M-Binding Buffer.Close the cap and mix by inverting the column several times.
?. [Bisulfite conversion]
Centrifuge at full speed (> 10,000 x g) for 30 seconds.Discard the flow-through.***REPEAT THE PREVIOUS STEP***
?. [Bisulfite conversion]
Add 100 μl of M-Wash Buffer to the column.Centrifuge at full speed for 30seconds.
?. [Bisulfite conversion]
Add 200 μl of L-Desulphonation Buffer to the column and let stand at room temperature (20-30°C) for 15-20 minutes.During this step, label Eppendorf Lo-Bind tubes in which the universal and sample index will be added (see below).After the incubation, centrifuge at full speed for 30 seconds.
?. [Bisulfite conversion]
Add 200 μl of M-Wash Buffer to the column. Centrifuge at full speed for 30 seconds.
?. [Bisulfite conversion]
Place the column into a LoBind Eppendorf tube (don't forget or you lost your sample) and add 15 μl of nuclease-free H2O (instead of the Elution buffer according to the protocol)* directly to the column matrix.Let stand at room temperature for 3 minutes. Centrifuge for 30 seconds at full speed to elute the DNA.The DNA is ready for immediate analysis or can be stored at or below -20°C for later use. For long-term storage, store at or below -70°C. *Elution buffer can inhibit the PCR.
?. [Bisulfite conversion]
Label new thin-walled Eppendorf tubes 0.2 mL: 2 per sample.As there is 50 µL total sample, two Eppendorf tubes per sample is needed.
?. [Bisulfite conversion]
***Repeat this wash step***
?. [Bisulfite conversion]
Label the columns.
?. [Final amplification step (KAPA HiFi HotStart Uracil+ ReadyMix PCR Kit (KAPA Biosystems)]
PCR protocol:5 minutes 95°C,17-19x (20 seconds 98°C, 15 seconds 65°C, 45 seconds 72°C)5 minutes 72°CMeanwhile, take the Ampure beads out of the fridge.
?. [Fragment Analyser]
Load samples onto the Fragment Analyser. Give additional PCR cycles based on the FA profile if neccesary.
?. [Magnetic bead purification]
Meanwhile, the sample is cleaned up by magnetic bead cleanup (Ampure)Label LoBind Eppendorf tubes:1 Eppendorf tube per sample for magnetic bead purification1 Eppendorf tube containing the final libraries: label carefully.Vortex the beads very thoroughly.Make FRESH 80% ethanol by adding 2 mL H2O to 8mL ethanol.
?. [Magnetic bead purification]
Add beads 2.5X the volume of the PCR (e.g. 30 µL PCR: add 75 µL beads)This removes all fragments
?. [Magnetic bead purification]
Incubate for 5 minutes.After this step, the beads contain DNA, so don't remove them.
?. [Magnetic bead purification]
Add Eppendorf tubes to magnet and incubate for 2 minutes.
?. [Magnetic bead purification]
Remove the liquid from the Eppendorf tubes.
?. [Magnetic bead purification]
Add 190 µL washing buffer (= 80% ethanol).
?. [Magnetic bead purification]
Remove 190 µL with 200 µL pipet, so no liquid remains.
?. [Magnetic bead purification]
***Repeat the washing step***
?. [Magnetic bead purification]
Remove any remaining ethanol with a smaller pipet.Dry the sample for 2-5 minutes at 38°C.
?. [Magnetic bead purification]
Remove the Eppendorf from the magnet.Add 20 µL TE buffer (0.1X) and pipet up and down
?. [Magnetic bead purification]
Incubate for 3 minutes.
?. [Magnetic bead purification]
DNA is now present in the TE buffer, so the beads can be removed.Pipet the mixture containing the sample and TE buffer to the final Eppendorf tube.Repeat the PCR if the libraries are not amplified enough (based on the FA results).