TITLE: GUIDE-seq simplified library preparation protocol (CRISPR/Cas9 off-target cleavage detection)
AUTHORS: Nagendra palani

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">GUIDE-seq is an experimental method to detect off-target cleavages caused during CRISPR/Cas editing.</div><div class = "text-block"><span>See publication </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1038/nbt.3117</span></a><span> for details about the method, the protocol used, and the analysis performed.</span></div><div class = "text-block">We identified several areas of improvement in the published protocol for GUIDE-seq NGS library preparation and developed a simplified protocol that is easier to perform & less expensive. The new protocol is also load-and-go compatible with standard sequencing runs on all Illumina instruments (using the kit SBS reagents & default instrument settings) compared to the NBT protocol that requires the libraries to be run on a MiSeq with some tweaks to the software configuration.</div><div class = "text-block">Notable changes:</div><div class = "text-block">1. Moved the UMI (Unique Molecular Identifier) from the Y-adapter to be inline with the insert. UMI is now sequenced as part of the sequencing read (R2 on a paired-end run). This eliminates the need to export index reads from the MiSeq.</div><div class = "text-block">2. Removed the sample index from the Y-adapter and changed the PCR scheme. Now, only a single adapter is required for ligation to any number of samples, PCR1 is for enrichment of the dsOligo - genome junction, PCR2 is to add sample-specific indexes to the enriched amplicons. </div><div class = "text-block">See oligo designs for further information.</div><div class = "text-block">3. Replaced sonication based shearing followed by end-repair with a one-pot, one-step enzymatic fragmentation + end-repair kit from New England Biolabs.</div></div>

[STEPS]
?. [Adaptor formation]
Use 0.1x TE to prepare 100 μM stock of all the oligos.In a 0.2 ml PCR tube, add the following reagents, vortex well, and spin down. Place on the thermal cycler at  for   (heated lid).    -------------------------------------------- After incubation, terminate the incubation and let the heat block cool down to room temperature. Remove the tube and place it on the bench for 1 hr.The oligos will have annealed and formed the adaptor.In a 0.2 ml PCR tube, add the following reagents, vortex well, and spin down. Place on the thermal cycler at for  (heated lid). --------------------------------------------After incubation, terminate the incubation and let the heat block cool down to room temperature. Remove the tube and place it on the bench for 1 hr.The oligos will have annealed and formed the adaptor.
[Oligo TA_Adaptor_Top (100 uM)]
[Oligo  TA_Adaptor_Bottom (100 uM)]
[dH2O]
[Total]
?. [DNA fragmentation, end-repair, ligation]
This section of the protocol is from the manual for NEB Ultra II FS kit - Catalog # E7805. (Refer to https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale7805.pdf : Section for Input > 100 ng for detailed protocol)Aliquot of genomic DNA into a well of a 96-well plate and use 1x TE to bring the volume to .Add the NEBNext Ultra II FS buffer + enzyme mix to the DNA (follow protocol from the linked manual)Set up the following program on the thermal cycler    for   for    hold. Add the NEBNext Ultra II ligation mix + enhancer to the well. Add  of the adaptor to the mixture. Mix well and incubate at   for  . Post incubation, add   of 0.1x TE and bring volume to  .Perform bead clean-up of the ligation mix using the sample purification beads that came with the kit. Set up the following program on the thermal cycler  for for  hold.Add the NEBNext Ultra II ligation mix + enhancer to the well. Add of the adaptor to the mixture. Mix well and incubate at  for . Post incubation, add  of 0.1x TE and bring volume to .Use  of beads for 1st bead addition of beads for 2nd bead addition (to select for an insert size distribution of 350 - 600 bp).Elute in of dH2O.
400 ng
26 µl
[heated lid]
This should fragment the library to have a distribution between 300 - 700 bp.
2 µl
[heated lid off]
28 µl
100 µl
20 µl
10 µl
17 µl
?. [Enrichment PCR]
Make 10 uM dilutions of the following primers.dsODN_Enrich_PlusdsODN_Enrich_MinusdsODN_Enrich_AdaptorFor each sample set up the following PCRs in a 96-well plate.PCR 1.1 - Plus_Strand_EnrichmentPCR 1.2 - Minus_Strand_EnrichmentPCR program:7 cycles13 cyclesAfter end of PCR, use of the PCR end-product for Picogreen / Qubit.Useto run the sample on a Tapestation (D5000 high-sensitivity) to check for library size.Each sample library should be a unimodal distribution between 400 - 1300 bp. All libraries should have an average size distribution within 15% across all samples.
[Adaptor ligated DNA]
[Ultra II Q5 master mix]
[dsODN_Enrich_Adaptor]
[dsODN_Enrich_Plus]
[Adaptor ligated DNA]
[Ultra II Q5 master mix]
[dsODN_Enrich_Adaptor]
[dsODN_Enrich_Minus]
[Initial denaturation]
[Denaturation]
[Gradient -1 ºC / cycle]
[Annealing]
[Extension]
[Denaturation]
[Annealing]
[Extension]
[Hold]
1 µl
2 µl
?. [Indexing PCR]
Make 10 uM dilutions of the Nextera_R1 & Nextera_R2 primers.For each sample, mix  of PCR 1.1  +of PCR 1.2Set up the following PCR: As required---------------------------------------------------PCR Program:10 cyclesAfter end of PCR, use of the PCR end-product for Picogreen / Qubit.
5 ng
5 ng
[Ultra II Q5 master mix]
[Nextera_R1]
[Nextera_R2]
[Template]
[dH2O]
[Total]
[Initial Denaturation]
[Denaturation]
[Annealing]
[Extension]
[Hold]
1 µl
?. [Library pooling, QC, Sequencing]
Pool samples by equal mass (assuming the library size distribution for the samples are similar). Do a 1.2x SPRI bead clean-up. Elute in  Check concentration of the pool by Picogreen/Qubit and quantify library using Kapa Illumina Library quantification kit. Dilute the pool to appropriate molarity required for the sequencing instrument.Load the pool on the sequencer and run with 15% PhiX spike-in for base diversity (works on MiSeq, should work with other Illumina 4-color instruments. Might need more PhiX for NextSeq.)Do a 1.2x SPRI bead clean-up. Elute in Check concentration of the pool by Picogreen/Qubit and quantify library using Kapa Illumina Library quantification kit. Dilute the pool to appropriate molarity required for the sequencing instrument.Load the pool on the sequencer and run with 15% PhiX spike-in for base diversity (works on MiSeq, should work with other Illumina 4-color instruments. Might need more PhiX for NextSeq.)
[0.1x TE]