TITLE: sci-ATAC-seq HTAN
AUTHORS: Andrew Adey, Andrew Fields, Casey Th, Ryan Mulqueen, Sarah Vitak, Eleonora Juarez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Single-cell combinatorial indexing ATAC-seq (Sci-ATAC-seq) workflow fro human cells/tissue.</div><div class = "text-block">This protocol is the up-to-date Adey Lab protocol that incorporates the use of Pitstop2</div></div>

[STEPS]
?. [Prepare Buffers and Pre-chill ]
Note: NIB keeps for ~1 month at 4C. For most preparations, 15 mL is sufficient.
?. [Nuclear Isolation]
Generalized nuclear isolation steps:1. Pellet  liquid cultured cells incentrifuge at 500 rcf  for 2. Aspirate pellet and resuspend in 3. Incubate on ice for3. Pellet liquid cultured cells incentrifuge at 500 rcf  for 4. Aspirate pellet and resuspend 5. Run total volume through 35 um filter 6. Add DAPI to final concentration of 15 ug/mL (ie for every 1 mL of sample).
NoteIsolation of nuclei is dependent on the sample being used. Tissue should follow a dounce homogenization protocol, while liquid cell cutures can be pelleted and resuspended directly in NIB.
10 ml
[min]
[NIB]
[min]
[min]
[NIB]
[5 mg/mL DAPI]
?. [Perform 1st sort]
1. Per well use and from Illumina2. Sort 2,000-5,0000 nuclei into each well using fuoresence activated cell sorting (FACS)
[NIB]
[TD buffer (2X)]
Nuclei count may vary by prep, but ensure that numbers within a prep are constant, ie all wells receive the same number of cells. Keep in ice or in 96-well plate chiller the whole time.
?. [Tagmentation following 1st Sort]
Spin down 8-well strips in table top centrifuge immediately after sorting finishes.	Store covered and on ice until the full plate is complete.	Note: to cover strips as they finish sorting, I tend to tear up aluminum plate covers into single-strip width.	Addition of Pitstop2 in 2X TD Buffer	Make sure to test the volume in a well following sort. Sorting a high count of nuclei (2-5K/well increases to volume by 5-10uL, respectively). If unadjusted, this will lower your TD buffer concentration and decrease Tn5 activity.	In 2X TD Buffer, add Pitstop2 reagent before addition into wells.	Example: Pitstop2 addition for a 5K nuclei sort (adding 10uL to each well which contains 10uL [NIB+2X TD Buffer] and 10uL of sorted nuclei; 30uL Final Volume).	Determining concentration of Pitstop2 for TD buffer addition:a.	C1V1=C2V2b.	(30uL Total Volume per well)(70uM Final Pitstop Conc.)=(10uL 2X TD Buffer)(X Pitstop2 Concentration)♣	X=210uMa.	Determining volume of Pitstop2 to add to 2X TD Buffer mastermix:♣	C1V1=C2V2♣	(210uM working concentration)(100uL TD mastermix) = (3000uM Pitstop2 stock)(X Pitstop2 volume)i.	X= 7uL Pitstop2 stock for each 93uL 2X TD buffer•	Add 1uL 8uM uniquely indexed transposome to each well•	Seal plate and incubate at 55C for 15 minutes with gentle shaking (on eppendorf thermomixer, ~300 rpm).•	Plate plate on ice immediately to stop reaction. Keep samples on ice to prevent over-transposistion and nuclei lysis.•	Pool all wells, while maintaining everything on ice.•	Add 2uL/per mL pooled sample of DAPI (5mg/mL) and bring to sorter for second sort.
?. [2nd Sort Plate Setup: Preparing Xie Buffer]
Preparing Second Plate Xie Buffer (8.5uL/well):VolumeReagent:6 mL0.05M Tris-HCl pH 7.8 40 uL0.5 EDTA200 uL1M KCl200 uL10% Triton X-100300 uLQiagen Protease3.26 mLH2O10 mLTotal•	Add 2.5 uL of 10 uM i5 Indexed PCR Primer and 2.5 uL of 10 uM i7 Indexed PCR Primer prior to sort.
?. [2nd Sort Protocol]
•	Thaw RT-PCR reagents on ice before second sort•	Sort X nuclei per well (X is dependent on number of wells tagmented in first sort, as a linear trend)•	1 plate = 22 nuclei/well•	1.5 plates = 33 nuclei/well•	2 plates = 44 nuclei/well etc...•	Using same gates as first sort:•	Sort X nuclei per well with modified sort settings:o	"Single cell" rather than "Normal"o	This leads to a higher abort count (less efficient sorting) but is far more precise•	As each 8-well strip (containing the Xie buffer) is completed, replace with a new prepared strip.•	Spin down the completed 8-well stip in table-top centrifuge beside the machine.•	Cover the strips and store on ice until all sorting is completed.•	Note: to cover strips as they finish sorting, I tend to tear up aluminum plate covers into single-strip width.•	Keep sorted samples on ice to prevent transposases cross-reacting with other nuclei.
?. [Transposase Denaturation and PCR setup]
•	Spin down 8-well strip tubes•	Cover plate and hold on ice until sorting is complete.•	Digest remaining Tn5 and then denature the Qiagen serine protease on Eppendorf Thermocyclers: •
?. [Transposase Denaturation and PCR setup]
Add 13.5 ul total of PCR Master Mix to each well:
[5X KAPA GC buffer]
[10 mM dntps]
[KAPA non-hotstart HiFi]
[100X SYBR Green I]
?. Perform PCR on the Biorad CFX Connect:Plate ReadPull once majority of wells begin to plateaus, for sciATAC libraries amplify between 14-19 cycles
?. [Library Clean-up and Quantification]
Pool 10uL for each well.	Ran full pool volume through Qiaquick PCR column following manufacturer's protocol.	Eluted in 32 uL 10mM Tris HCl pH 8	Quantified 2 uL in 2:200 dsDNA HS Qubit assay•	This is to ensure that the library amount will be visible on a gel.Use Qubit reading to dilute to ~4ng/uL and run 1uL on HS Bioanalyzer chipo	Run 1uL of sample at 4ng/uL library dilution on Bioanalyzer High-Sensitivity DNA chip (following manufacturer's protocol)o	Quantify library from the range of 100-1000bpo	Dilute this down to 1nM concentration for sequencing.