TITLE: scTHS-seq 
AUTHORS: Kun Zhang, Brandon Sos, Dinh Diep, Thu E Duong

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Single cell transposome hypersensitive site sequencing (scTHS-seq) combined transposome hypersensitive site sequencing (THS-seq) with combinatorial cellular indexing using customized barcoded transposomes. Compared to a similar method, ATAC-seq, THS-seq have improved coverage of distal enhancer sites which is due to the utilization of an  engineered super-mutant Tn5 transposase as well as in vitro transcription to perform linear amplification from a single insertion site of the Tn5 transposase instead of two insertion sites required in ATAC-seq. scTHS-seq was used to generate single nucleus accessibility profile of 36,869 cells with an average of 10,168 unique reads per cell that are also associated with a predicted accessible region. The doublet rate of scTHS-seq is estimated to be between 20-25% which is comparable to similar combinatorial indexing methods.</div></div>

[STEPS]
?. [Tagmentation]
Generate 384 annealed transposonsCombine top and bottom strand oligos in a PCR tube. In a thermocycler, incubate for 2 minutes at  then ramp to at a rate of  per second.
[Indexed Tn5 top strand (100uM)]
[5' phosphorylated mosaic end sequence bottom strand (100uM)]
?. [Tagmentation]
Load Tn5-059
[4.8 uM transposase Tn5-059]
[diluted annealed transposons (6.5 uM)]
?. [Tagmentation]
Wash nuclei or cells suspension with ice-cold PBS
?. [Tagmentation]
Resuspend 400,000 cells/nuclei in lysis buffer. Use 100 uL per 100,000 cells/nuclei
?. [Tagmentation]
Resuspend nuclei in 100 uL 1.5X D6 buffer initially. Count and add more 1.5X D6 buffer to create ~250 nuclei/uL
?. [Tagmentation]
Aliquot 4.0 uL of cells in 1.5X D6 buffer into wells containing 2.0 uL of prepared transposomes
?. [Tagmentation]
Incubation without shaking
[Thermocycler]
?. [Tagmentation]
Terminate the tagmentation reaction with EDTA
[EDTA, 50 mM]
?. [Tagmentation]
Store cells at -20C overnight or until ready to sort
?. [FACS (BD Influx)]
Coat 12 x 200  uL, 1 x 15 mL conical using 1X FACS buffer
[Let tubes sit in FACS buffer]
?. [FACS (BD Influx)]
Add 1 volume (10 uL) ice cold 2X FACS buffer to each sample well with cells in tagmentation mix
?. [Tagmentation]
Make 1000 uL transposase dilution buffer
[75% glycerol/0.3% TritonX-100]
[DTT, 1M]
[Tris-HCl, 4M, pH 7.5]
[nuclease free water]
?. [Tagmentation]
Make 10 mL 75% glycerol with 0.3% tritonX-100
[Glycerol (100%)]
[TritonX-100, 10%]
[nuclease free water]
?. [Tagmentation]
Make 10 mL of 1.5X D6 buffer
[Tango Buffer, 10X]
[DMF, 100%]
[nuclease free water]
?. [Tagmentation]
Make 10 mL of 1X lysis bufferFinally, add 1 protease inhibitor tablet (EDTA-free).
[Tris-HCl, 4M, pH 7.5]
[Sodium chloride, 5M]
[Magnesium chloride, 2M]
[IGEPAL-630, 10%]
[BSA, 10%]
[PBS, 10X]
[nuclease free water]
?. [Tagmentation]
Make 10 mL of 2X FACS buffer
[PBS, 10X]
[BSA, 10%]
[EDTA, 0.5M]
[nuclease free water]
?. [Tagmentation]
Dilute annealed transposons
[Annealed transposons]
[transposon dilution buffer]
?. [Tagmentation]
Make 420 uL of 4.8 uM transposase Tn5-059
[transposase, Tn5-059, 8 uM]
[transposase dilution buffer]
?. [Tagmentation]
Incubate
[Thermocycler]
?. [Tagmentation]
Incubate
[On ice]
?. [Tagmentation]
Pellet by centrifuging at 900 RCF
?. [Tagmentation]
Count cells
?. [Tagmentation]
Pellet 400,000 cells / nuclei by centrifuging at 330 RCF (cells) or 900 RCF (nuclei)
?. [Tagmentation]
Incubate
[Thermocycler]
?. [FACS (BD Influx)]
When 15 mL conical have been coated for 1 hr, pool all samples together using multichannel pipette and adding to 12 x 200 uL tubes
?. [FACS (BD Influx)]
Centrifuge at 500 RCF
?. [FACS (BD Influx)]
Resuspend nuclei in 1.5 mL 1X FACS buffer
?. [FACS (BD Influx)]
Add stain for sorting
[Propidium Iodide]
?. [FACS (BD Influx)]
Sort 100 cells into each well using BD Influx, remove doublets using forward and side scatter plots and selecting for PI-staining events
?. [FACS (BD Influx)]
Add 10 uL PBS to each well of 10 to 15 x 96-wells plate for sorting
Cover and store all plates on ice.
?. [In vitro transcription (10 plates of sorted nuclei)]
Add 11 uL of 8M guanidine chloride to each well using multi-channel to lyse nuclei and release DNA
[Guanidine hydrochloride, 8M]
?. [In vitro transcription (10 plates of sorted nuclei)]
Add 30 uL of KAPA Pure Beads to each well to bind DNA
[KAPA Pure Beads]
?. [In vitro transcription (10 plates of sorted nuclei)]
Mix well and bind for at least 8 minutes
[Room temperature]
?. [In vitro transcription (10 plates of sorted nuclei)]
Place plate on magnet for at least 3 minutes then discard the supernatant
[Room temperature]
?. [In vitro transcription (10 plates of sorted nuclei)]
Prepare fresh 80% ethanol solution. Wash beads (while still on magnet) twice with 170 uL of 80% ethanol
[Freshly prepared 80% ethanol]
?. [In vitro transcription (10 plates of sorted nuclei)]
After last wash, completely remove all ethanol and transfer to a heated thermomixer to dry
?. [In vitro transcription (10 plates of sorted nuclei)]
Add 8 uL of nuclease free water to each well using a multichannel pipette
[nuclease-free water]
?. [In vitro transcription (10 plates of sorted nuclei)]
Add 2 uL of 5X Taq Master Mix to perform gap filling
[5X Taq Master Mix]
?. [In vitro transcription (10 plates of sorted nuclei)]
Incubate plates for gap filling
?. [In vitro transcription (10 plates of sorted nuclei)]
Add 2 uL of HiScribe T7 IVT reaction components to each well
[T7 buffer, 10X]
[UTP]
[GTP]
[ATP]
[CTP]
[T7 RNA Polymerase]
?. [In vitro transcription (10 plates of sorted nuclei)]
Incubate
[Thermocycler]
?. [In vitro transcription (10 plates of sorted nuclei)]
Add 40 uL of KAPA Pure Beads to each well to bind DNA
[KAPA Pure Beads]
?. [In vitro transcription (10 plates of sorted nuclei)]
Mix well and bind for at least 8 minutes
[Room temperature]
?. [In vitro transcription (10 plates of sorted nuclei)]
Place plate on magnet for at least 3 minutes then discard the supernatant
[Room temperature]
?. [In vitro transcription (10 plates of sorted nuclei)]
Prepare fresh 80% ethanol solution. Wash beads (while still on magnet) twice with 170 uL of 80% ethanol
[Freshly prepared 80% ethanol]
?. [In vitro transcription (10 plates of sorted nuclei)]
After last wash, completely remove all ethanol and transfer to a heated thermomixer to dry
?. [In vitro transcription (10 plates of sorted nuclei)]
Add 9 uL of nuclease free water to each well using a multichannel pipette
[nuclease-free water]
?. [cDNA synthesis (10 plates of sorted nuclei)]
Add 2.5 uL of random hexamers
[random hexamers, 20 uM]
?. [cDNA synthesis (10 plates of sorted nuclei)]
Incubate
[Thermocycler]
Cool immediately on ice after.
?. [cDNA synthesis (10 plates of sorted nuclei)]
Add 9.5 uL of MMLV RT mixture
[First strand buffer, 5X]
[Advantage UltraPure dNTPs, 100 uM each]
[DTT, 100mM]
[MMLV RT]
?. [cDNA synthesis (10 plates of sorted nuclei)]
Incubate in a thermocycler
?. [cDNA synthesis (10 plates of sorted nuclei)]
Add 1 uL RNase H
[nuclease-free water]
[RNase H, 5 Units/uL]
?. [cDNA synthesis (10 plates of sorted nuclei)]
Incubate in thermocycler
?. [cDNA synthesis (10 plates of sorted nuclei)]
Add 2.5 uL second strand synthesis primer
[sss_scnXTv2, 20 uM]
?. [cDNA synthesis (10 plates of sorted nuclei)]
Incubate in thermocycler
Cool immediately on ice after incubation
?. [cDNA synthesis (10 plates of sorted nuclei)]
Add 5.9 uL 5X Taq Master Mix
[Taq Master Mix, 5X]
?. [cDNA synthesis (10 plates of sorted nuclei)]
Incubate in thermocycler
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Add 40 uL of KAPA Pure Beads to each well to bind DNA
[KAPA Pure Beads]
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Mix well and bind for at least 8 minutes
[Room temperature]
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Place plate on magnet for at least 3 minutes then discard the supernatant
[Room temperature]
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Prepare fresh 80% ethanol solution. Wash beads (while still on magnet) twice with 170 uL of 80% ethanol
[Freshly prepared 80% ethanol]
?. [Tagmentation on beads (10 plates of sorted nuclei)]
After last wash, completely remove all ethanol and transfer to a heated thermomixer to dry
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Add 4 uL of 1.5X tagmentation buffer to each well using a multichannel pipette
[nuclease-free water]
[Tango Buffer, 10X]
[DMF, 100%]
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Generate annealed transposonsCombine top and bottom strand oligos in a PCR tube. In a thermocycler, incubate for 2 minutes at  then ramp to at a rate of  per second.
[i7 top strand (100uM)]
[5' phosphorylated mosaic end sequence bottom strand (100uM)]
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Dilute annealed transposons
[Annealed transposons]
[transposon dilution buffer]
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Make 420 uL of 2.4 uM transposase Tn5-059
[transposase, Tn5-059, 8 uM]
[transposase dilution buffer]
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Load Tn5-059
[2.4 uM transposase Tn5-059]
[diluted annealed transposons (6.5 uM)]
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Incubate
[Thermocycler]
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Add 2 uL of loaded transposomes to each well
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Incubate
[Thermocycler]
Cool immediately on ice after.
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Add 6 uL guanidine hydrochloride
[Guanidine hydrochloride, 8M]
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Add 30 uL of KAPA Pure Beads to each well to bind DNA
[KAPA Pure Beads]
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Mix well and bind for at least 8 minutes
[Room temperature]
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Place plate on magnet for at least 3 minutes then discard the supernatant
[Room temperature]
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Prepare fresh 80% ethanol solution. Wash beads (while still on magnet) twice with 170 uL of 80% ethanol
[Freshly prepared 80% ethanol]
?. [Tagmentation on beads (10 plates of sorted nuclei)]
After last wash, completely remove all ethanol and transfer to a heated thermomixer to dry
?. [Tagmentation on beads (10 plates of sorted nuclei)]
Add 16 uL nuclease free water
[nuclease-free water]
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
Add 12 uL of qPCR mix to each well of a new qPCR plate
[(activated) KAPA HiFi HotStart Ready Mix, 2X]
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
Make a strip tube of 16 different s5XX primers (5XX is a different primer id). Aliquot 2 uL to each well along the rows of each plate
[s5XX primer, 5 uM]
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
Make a strip tube of 12 different i7XX primers (7XX is a different primer id). Aliquot 2 uL to each well along the columns of each plate
[i7XX primer, 5 uM]
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
Perform amplification using qPCR with plate read after the extension stepThen 6 cycles of the following:Switch to 2 steps PCR for 3-4 cycles to enrich for fully adapted products:Perform final extension step
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
Pool 2 uL of each sample together, keeping each plate a separate pool
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
Add 450 uL of KAPA Pure Beads to each pool to bind DNA
[KAPA Pure Beads]
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
Mix well and bind for at least 8 minutes
[Room temperature]
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
Place plate on magnet for at least 3 minutes then discard the supernatant
[Room temperature]
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
Prepare fresh 80% ethanol solution. Wash beads (while still on magnet) twice with 170 uL of 80% ethanol
[Freshly prepared 80% ethanol]
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
After last wash, completely remove all ethanol and transfer to a heated thermomixer to dry
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
Add 30 uL nuclease free water
[nuclease-free water]
Libraries must be size selected (for 220-1000 bp) with polyacrylamide or agarose gels before sequencing to avoid cross contamination due to leftover primers
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
Add nuclease free water to make the volume in each pool 250 uL total
?. [Tagmentation]
Make 3.045 mL transposons dilution buffer
[75% glycerol/0.3% TritonX-100]
[Qiagen EB]
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
Incubate hotstart polymerase master mix to activate the enzyme
[KAPA HiFi Hotstart Ready Mix, 2X]
[Thermocycler]
?. [Test amplification (10 plates of sorted nuclei)]
Preheat hotstart polymerase mix
[KAPA HiFi Hotstart ReadyMix, 2X]
[Thermocycler]
Cool immediately on ice
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
Transfer 8 uL of elution from sample plates to the new plates
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
?. [Amplification with adapter primers (10 plates of sorted nuclei)]
?. [Test amplification (10 plates of sorted nuclei)]
Add primers to make a master mix to 417 nM final each
[i5XX, 5 uM]
[i7XX, 5 uM]
?. [Test amplification (10 plates of sorted nuclei)]
Add SYBR Green for qPCR to 0.8X final
[SYBR Green, 100X]
?. [Test amplification (10 plates of sorted nuclei)]
Aliquot 8 uL of amplification mix to each well to qPCR plate
?. [Test amplification (10 plates of sorted nuclei)]
Sample 5 wells from each plate, adding 4 uL of each sample. Include an no nuclei control well.
?. [Test amplification (10 plates of sorted nuclei)]
Perform qPCR. Monitor the sample progress, terminate when all samples appears to be in exponential phase (usually 6-8 cycles). No nuclei control may appear to amplify but majority of those products are primer dimers.1. 72C x 5 min2. 98C x 30 s3. 59C x 30 s4. 72C x 1 minPlate read5. Go to 2 for 20 cycles6. 72C for 5 min
?. [Test amplification (10 plates of sorted nuclei)]
Assess PCR products on a 6% TBE PAGE gel. If lack of products, repeat test amplification on a different sampling of wells but adding 3-4 more cycles of amplification. If samples appear to be over-amplified, reduce the number of cycles by 1.
?. [Library amplification (10 plates of sorted nuclei)]
Preheat hotstart polymerase mix
[KAPA HiFi Hotstart ReadyMix, 2X]
[Thermocycler]
Cool immediately on ice
?. [Library amplification (10 plates of sorted nuclei)]
Aliquot 12 uL of preheated KAPA HiFi ReadyMix to each well of a new PCR plate
?. [Library amplification (10 plates of sorted nuclei)]
Aliquot 2 uL of 16 different s5XX primers to each set of 2 plates (one per row)
[each s5XX primer, 5 uM]
?. [Library amplification (10 plates of sorted nuclei)]
Aliquot 2 uL of 12 different s7XX primers to each column of every plate (one per column)
[s7XX primer, 5 uM]
?. [Library amplification (10 plates of sorted nuclei)]
Transfer 8 uL of sample to each well of PCR plate
?. [Library amplification (10 plates of sorted nuclei)]
Perform PCR using the number of cycles determined previously with test PCR1. 72C x 5 min2. 98C x 30 s3. 59C x 30 s4. 72C x 1 min5. Go to 2 for XX cycles6. 72C for 5 min
?. [Library amplification (10 plates of sorted nuclei)]
Pool 4 uL of each well from 2 plates with non-overlapping s5XX primers. Final volume is ~800 uL per pool. 5 pools total
?. [Library amplification (10 plates of sorted nuclei)]
Add 800 uL of KAPA Pure Beads to each pool to bind DNA
[KAPA Pure Beads]
?. [Library amplification (10 plates of sorted nuclei)]
Mix well and bind for at least 8 minutes
[Room temperature]
?. [Library amplification (10 plates of sorted nuclei)]
Place plate on magnet for at least 3 minutes then discard the supernatant
[Room temperature]
?. [Library amplification (10 plates of sorted nuclei)]
Prepare fresh 80% ethanol solution. Wash beads (while still on magnet) twice with 1000 uL of 80% ethanol
[Freshly prepared 80% ethanol]
?. [Library amplification (10 plates of sorted nuclei)]
After last wash, completely remove all ethanol and transfer to a heated thermomixer to dry
?. [Library amplification (10 plates of sorted nuclei)]
Add 20 uL of nuclease free water to each pool
[nuclease-free water]
?. [Library amplification (10 plates of sorted nuclei)]
Quantify each pool using dsDNA HS Qubit
?. [Library amplification (10 plates of sorted nuclei)]
Perform gel size selection for 220-1000 bp using 6% TBE PAGE. Use 5-wells gel, and loading ~1 ug of library per well.