TITLE: Loading Tn5 Enzyme with sci-protocol Oligonucleotides
AUTHORS: Ryan     Mulqueen, Andy Fields, Andrew    Adey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Loading of Tn5 enzyme for sci-ATAC and s3 protocols.</div></div>

[STEPS]
?. [Prepare Reagents]
Prepare   for protein dilution.   ABCD1ReagentStock ConcentrationFinal ConcentrationAmount of Stock2HEPES-KOH (pH 7.2)1M100mM5mL3NaCl5M200mM2mL4Glycerol100%25%12.5mL5Triton-X100100%0.2%100uL6ddH2O30.4mL (to 50mL)7DTTDry2mM15.4 mg
[2.125X Tn5 Dilution buffer]
ABCD1ReagentStock ConcentrationFinal ConcentrationAmount of Stock2HEPES-KOH (pH 7.2)1M100mM5mL3NaCl5M200mM2mL4Glycerol100%25%12.5mL5Triton-X100100%0.2%100uL6ddH2O30.4mL (to 50mL)7DTTDry2mM15.4 mg
?. [Prepare Reagents]
Prepare Mosaic End reverse compliment (ME'), i7,  i5 oligonucleotides at See attached spreadsheet for oligonucleotide sequences. Three sets of oligonucleotides are listed for both i5 and i7 Tn5 loading. This yields (3 i5 sets) x (3 i7 sets)=9 uniquely identifiable 96 well plates or 864 unique well barcode combinations.Mosaic End oligonucleotide sequence used for Tn5 loading is also listed within the spreadsheet.
[Tris-HCl buffer (pH 8.0)]
Synthesis quality of these oligonucleotides is critical. HPLC purification is essential. We find that Eurofins oligos outperform IDT by roughly 10 fold in library complexity.
All indexes are designed to be 2 or greater Hamming distance from all others to allow for sequencing errors.
?. [Anneal Indexed Oligoes to Mosaic End Reverse Compliment]
Preparation of dsDNA through annealing.Volumes are adjusted for a single 96-well plate loading.  AB112.5 uL100 uM i5 Tn5 Indexed oligo212.5 uL100 uM Mosaic End Reverse Compliment oligo353.125 uL2.125x Tn5 Dilution Buffer2. For each i7 barcoded oligo prepare the following reaction (12 total):  AB18.5 uL100 uM i7 Tn5 Indexed oligo28.5 uL100 uM Mosaic End Reverse Compliment oligo336.125 uL2.125x Tn5 Dilution Buffer
AB112.5 uL100 uM i5 Tn5 Indexed oligo212.5 uL100 uM Mosaic End Reverse Compliment oligo353.125 uL2.125x Tn5 Dilution Buffer
AB18.5 uL100 uM i7 Tn5 Indexed oligo28.5 uL100 uM Mosaic End Reverse Compliment oligo336.125 uL2.125x Tn5 Dilution Buffer
?. [Anneal Indexed Oligoes to Mosaic End Reverse Compliment]
Anneal Oligo mixtures within a Thermocycler with the following reaction.Slow ramp down to This results in annealed oligo species per reaction (i7/ME' and i5/ME').
95 °C
[at a rate of -2.5C/min]
[hold]
Oligoes should be freshly annealed prior to loading Tn5 transposome
?. [Plate Annealed Oligos]
Prepare a 96-well plate with the following loading schema.1. Add  of i5/ME' () to each respective wells in a row-wise fashion.2. Add  of i7/ME' () to each respective wells in a column-wise fasion.This results in  at /well
5 µl
5 µl
[i5/ME' and i7/ME' Indexed Oligos]
?. [Adjust Salt Concentration on Tn5 and Load]
Prepare Tn5 protein as described in "Generation and Purification of pTXB1.Tn5" protocol.Prior to loading Tn5 protein adjust NaCl concentration.Combine:  AB11152 uLPrepared Tn52144 uL5M NaCl
AB11152 uLPrepared Tn52144 uL5M NaCl
?. [Adjust Salt Concentration on Tn5 and Load]
Add  of salt-corrected Tn5 to each well of the 96 well plate.Assemble the Tn5/oligo mixture via incubation at  for  .
12 µl
25 °C
Store at -20C for no more than 8 months.