TITLE: ATAC-STARR-seq
AUTHORS: Tyler  Hansen, Emily Hodges

[DESCRIPTION]
Massively parallel reporter assays test the capacity of putative cis-regulatory elements (CREs) to drive transcription on a genome-wide scale. In nearly all cases, chromatin accessibility is necessary to drive activity, so most CREs are inactive due to chromatin context rather than intrinsic DNA sequence properties. Here, we combined assay for transposase-accessible chromatin (ATAC-seq) with self-transcribing active regulatory region sequencing (STARR-seq) to selectively assay the regulatory potential of nucleosome-free DNA genome-wide. Our approach enabled high-resolution testing of ~50 million unique DNA fragments tiling ~101,000 accessible chromatin regions in human lymphoblastoid cells. To illustrate the application of our approach, we show that 30% of all accessible regions contain an activator, a silencer or both. Benchmarking against standard ATAC-seq, our approach faithfully captures chromatin accessibility and transcription factor (TF) footprints with high signal-to-noise. Integrating three layers of genomic information (accessibility, TF occupancy, and activity) provided by ATAC-STARR-seq, we stratified active and silent CREs by the presence of several TF footprints and show that CREs with specific TF combinations are associated with distinct gene regulatory pathways. Altogether, these data highlight the power of ATAC-STARR-seq to comprehensively investigate the regulatory landscape of the human genome from a single DNA source.

[STEPS]
SECTION: Anneling Oligos
1. Dilute oligos to 100 µM in STE buffer.
SECTION: Anneling Oligos
2. Make Adaptor Mixes in 200μL PCR tubes:

Mix (Adaptor A) [50µL]: 
    5µL Tn5MERV oligo (100µM)
    5µL Tn5_1 Oligo (100µM)
    40µL H2O

Mix (Adaptor B) [50µL]: 
    5µL Tn5MERV oligo (100µM)
    5µL Tn5_2_ME_Comp Oligo (100µM)
    40µL H2O
SECTION: Anneling Oligos
3. Anneal Adaptor solutions separately with thermocycler conditions as follows:
95°C, 3 min
65°C, 3 min
Ramp to 24°C, -1ºC per cycle, 30 s
Hold at 24°C forever
SECTION: Anneling Oligos
4. After annealing mix: 
50µL Adaptor solution A
50µL Adaptor solution B
100µL Glycerol (≥99.5%)
200 µL Adaptor mixture, which is now 5 µM.
SECTION: Transposome Assembly
5. Mix the following:
10 µL 5µM Adaptors Mix
10 µL Tn5 (3mg/mL in Tn5 storage buffer)
20 µL Total Reaction Volume

SECTION: Transposome Assembly
6. Let stand at room temp for 30-60min, then immediately place on ice. Assembled transposome may be stored at -20°C for several months. 
SECTION: ATAC Reaction
7. Turn on all equipment! 

Set the microcentrifuge to 4ºC & the thermomixer to 37ºC. Each of these takes 15-30min to get to their desired temp.

SECTION: ATAC Reaction
8. Count cells in duplicate using a hemocytometer. Record cell count. 

Note: It is important that cell viability is greater than 85%. If less than 85%, I strongly recommend trying again when viability is much better.
SECTION: ATAC Reaction
9. In two tubes, pellet 200,000 viable cells in each tube at 500 RCF at 4°C for 10 min in a fixed angle centrifuge. 

Use low-bind Eppendorf tubes to prevent pellets from sticking along the back of the tube (this is much more important for reproducibility than you might think).
SECTION: ATAC Reaction
10. While waiting, make lysis buffers.
SECTION: ATAC Reaction
10.1. ATAC-Resuspension Buffer (RSB) containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin (lysis buffer):

For 110μL, mix 1.1μL 10% Tween-20,1.1μL 10% IGEPAL CA-630, 1.1μL 1% digitonin, 106.7μL 1x RSB.
SECTION: ATAC Reaction
10.2. ATAC-RSB containing only 0.1% Tween-20 (wash out buffer):

For 2.2mL, mix 220μL 10% Tween-20 and 2mL 1x RSB.

Keep on ice.
SECTION: ATAC Reaction
11. Aspirate supernatant and resuspend each cell pellet in 50µL cold ATAC-Resuspension Buffer (RSB) containing 0.1% IGEPAL CA-630, 0.1% Tween-20, and 0.01% Digitonin and pipette up and down 5 times. 

Note: The volume used here is a modification to the Omin-ATAC protocol. In my hands, 50μL for this number of cells works just as well as 200μL and is much more manageable.
SECTION: ATAC Reaction
12. Incubate on ice for 3 minutes.
SECTION: ATAC Reaction
13. Wash out lysis with 1 ml of cold ATAC-RSB containing ONLY 0.1% Tween-20 and invert tube 3 times to mix. 

Note: The volume used here is a modification to the Omin-ATAC protocol. In my hands, 1mL for this number of cells works just as well as 4mL and is much more manageable.
SECTION: ATAC Reaction
14. Immediately pellet nuclei at 500 RCF for 10min at 4°C in a fixed angle centrifuge. While waiting, make transposition mix:  
  
SECTION: ATAC Reaction
15. Aspirate all supernatant, carefully avoiding visible cell pellet, using two pipetting steps (aspirate down to 100 ul with a p1000 pipette and remove final 100 ul with a p200 pipette).
SECTION: ATAC Reaction
16. Resuspend cell pellets in 180µL of transposition mix by pipetting up and down 10 times. Combine like samples for a total volume of 360μL. 
SECTION: ATAC Reaction
17. Add 40µL assembled Tn5 transposase and quickly vortex to mix well. This is the equivalent to 5μL/reaction. 
SECTION: ATAC Reaction
18. Divide resuspension into 8-1.5mL tubes (50μL/tube).
SECTION: ATAC Reaction
19. Incubate reaction at 37°C for 30min in a thermomixer with 1000 RPM mixing.
SECTION: ATAC Reaction
20. After incubation, pool the eight like-reactions together in a 15mL conical tube containing 2mL DNA binding Buffer. 
SECTION: ATAC Reaction
21. Purify the small-isolated DNA fragments using the Zymo C+C kit. Run all 2.4mL through one column, adding in 800μL increments. Elute in 22µl pre-warmed elution buffer. Incubate on column for 5min. 

Can stop here and store at -20°C. 
SECTION: PCR Amplification
22. Make PCR mastermix: 

Note: do not use a hot-start polymerase here. The reaction requires polymerase activity in the initial extension step. 

  
SECTION: PCR Amplification
23. Run PCR with the following thermocycler parameters:

72 ºC, 5 min
98ºC, 30 sec
4 cycles of
98ºC, 10 sec
62ºC, 30 sec
72ºC, 1 min
Final extension 72ºC, 2 min
SECTION: PCR Amplification
24. Purify PCR Product using Zymo Clean and Concentrator Kit. Mix 250μL Binding Buffer with 50μL PCR products. Elute in 32µl pre-warmed elution buffer. Incubate on column for 5min. 
SECTION: PCR Amplification
25. Analyze by running 2uL on High-Sensitivity D5000 Tapestation. Determine size distribution and molarity. 

If it looks good, move on to cloning. Need between 0.04pmols and 0.27pmols total—no more, no less. If there are too many large fragments, perform the SPRI bead size selection below. 
SECTION: SPRI bead size-selection
26. *Note: Perform a 0.6X ratio right-side selection to remove fragments larger than 500bp. The volumes listed below are for that ratio specifically. Reference the SPRISelect User Manual if this needs to be altered. 

Thoroughly shake the SPRIselect bottle to resuspend the SPRI beads. 
SECTION: SPRI bead size-selection
27. Add 18μL of SPRIselect to the sample. Pipette up and down and vortex for at least 5 seconds to mix thoroughly.

Note: Insufficient mixing of sample and SPRIselect will lead to inconsistent size selection results.
SECTION: SPRI bead size-selection
28. Place the reaction vessel on DynaMag-PCR magnet and allow the SPRI beads to settle to the magnet for ~1min.
SECTION: SPRI bead size-selection
29. Transfer the clear supernatant, which contains the Right-Side Size Selected sample, to a new reaction vessel. The reaction vessel with the remaining beads can be discarded.
SECTION: SPRI bead size-selection
30. Add 36μL of SPRIselect to the Right-Side Size Selected sample. Pipette up and down and vortex for at least 5 seconds to mix thoroughly.
SECTION: SPRI bead size-selection
31. Place the reaction vessel on an appropriate magnetic stand or plate and allow the SPRI beads to settle to the magnet for ~1min. 
SECTION: SPRI bead size-selection
32. Remove and discard the clear supernatant. 
SECTION: SPRI bead size-selection
33. With the reaction vessel still on the magnet, add 180 μL of freshly-made 85% ethanol and incubate at RT for 30 seconds. Remove and discard ALL of the ethanol supernatant. 
SECTION: SPRI bead size-selection
34. Let the beads dry but not over dry. 
SECTION: SPRI bead size-selection
35. Once cracks are starting to form in the pellet, elute by removing the reaction vessel from the magnet and adding 12μL of 10mM Tris-HCl pH 8.0, elution buffer, or nuclease-free water.
SECTION: SPRI bead size-selection
36. Pipette up and down and vortex for at least 5 seconds to mix thoroughly.
SECTION: SPRI bead size-selection
37. Place the reaction vessel on an appropriate magnetic stand or plate and allow the SPRI beads to settle to the magnet for ~1min. 
SECTION: SPRI bead size-selection
38. Transfer the eluate (size selected sample) to an appropriate storage vessel. 

Note: It is very important to avoid pipetting beads over to the new tube. They can cause the tape station to run inaccurately. 
SECTION: SPRI bead size-selection
39. Analyze size-selected sample by running on 2uL on High-Sensitivity D5000 Tapestation and analyze for concentration and size range. 
SECTION: Linearization of hSTARR-Seq_ORI
40. *Note: at this step, you will generate the vector required for the gibson cloning reaction. This linearized vector contains the homology arms for gibson. In the NAR paper we used an oligo to add the N504 barcode to the plasmid, this is not required for most purposes. For this reason, I’ve denoted the reverse primer as N5XX/universal because you can add whatever index you want OR not add an index at all, just like the forward primer. 


PCR amplify vector backbone from the hSTARR-Seq_ORI plasmid (Addgene #99296) with the mixture and thermocycler conditions below. 
  
98ºC, 30 sec
2 cycles of
98ºC, 10 sec
59ºC, 30 sec
72ºC, 90 sec
28 cycles of
98ºC, 10 sec
72ºC, 2min
Final extension 72ºC, 2 min
SECTION: Linearization of hSTARR-Seq_ORI
41. Purify PCR Product using Zymo Clean and Concentrator – 5 Kit. Elute in 20μL elution buffer.
SECTION: Linearization of hSTARR-Seq_ORI
42. Determine concentration via Nanodrop. 
SECTION: Linearization of hSTARR-Seq_ORI
43. Analyze by running 0.1ng on 1% agarose gel with EtBr stain to determine purity. 
SECTION: Clone fragments into linearized hSTARR-Seq_ORI via Gibson Cloning
44. *This part of the protocol is intentionally flexible in order to add the entire volume of tagments to these gibson reactions. In this step, perform four Gibson reactions per sample at a 1:2 rato of vector:insert. 

Note: this used to be a 1:3 ratio but was changed to 1:2 after learning that 1:2 would require fewer PCR cycles and is the recommended ratio for this enzyme cocktail. 

The insert (tagments) should be at a total amount of 0.04pmols and 0.27pmols. Calculate the reaction molarity as follows:

  
SECTION: Clone fragments into linearized hSTARR-Seq_ORI via Gibson Cloning
45. In 4 PCR tubes, combine the volumes of insert and vector as written above. 
SECTION: Clone fragments into linearized hSTARR-Seq_ORI via Gibson Cloning
46. Bring up to a total volume of 5μL with water. 

Note: The total volume cannot exceed 5μL.
SECTION: Clone fragments into linearized hSTARR-Seq_ORI via Gibson Cloning
47. In a separate PCR tube, preparea negative control where you substitute the inserts for water (only 1 reaction). 
SECTION: Clone fragments into linearized hSTARR-Seq_ORI via Gibson Cloning
48. Add 5μL of NEBuilder Hifi DNA Assembly 2x MasterMix and mix well.
SECTION: Clone fragments into linearized hSTARR-Seq_ORI via Gibson Cloning
49. Incubate at 50°C for 1hr. 
SECTION: Ethanol Precipitation of Gibson Reactions
50. *This is needed to prevent arching in the Electroporation step. DO NOT SKIP. 

Pool all four like-reactions together into a low-bind Eppendorf tube. 
SECTION: Ethanol Precipitation of Gibson Reactions
51. Adjust volume to 222.5μL by adding 182.5μL elution buffer (212.5μL to negative control).
SECTION: Ethanol Precipitation of Gibson Reactions
52. Add 25μL 3M NaAc pH5.2 and Vortex
SECTION: Ethanol Precipitation of Gibson Reactions
53. Add 2.5μL GlycoBlue and Vortex. 
SECTION: Ethanol Precipitation of Gibson Reactions
54. Add 750μL cold (-20°C) 100% EtOH and vortex. 
SECTION: Ethanol Precipitation of Gibson Reactions
55. Incubate at -20°C for 30-120min. 
SECTION: Ethanol Precipitation of Gibson Reactions
56. Spin at full speed for 30min @ 4°C
SECTION: Ethanol Precipitation of Gibson Reactions
57. Aspirate supernatant
SECTION: Ethanol Precipitation of Gibson Reactions
58. Wash with 750μL cold 70% EtOH. Spin at full speed for 15min @ 4°C. 
SECTION: Ethanol Precipitation of Gibson Reactions
59. Aspirate supernatant and repeat wash. 
SECTION: Ethanol Precipitation of Gibson Reactions
60. Dry pellet at room temp until dry. 
SECTION: Ethanol Precipitation of Gibson Reactions
61. Resuspend pellet in 10μL 10mM Tris-HCl pH 8.0. Pipette up/down until reconstituted. Can stop here, store @ -20°C. 
SECTION: Electroporation of STARR-seq Plasmid Library into Bacterial Cells
62. *In addition to the negative control, do a positive control using the supplied pUC19. For the controls, only do one reaction and use refrozen cells if possible. 

Pre-cool pipette tips, GenePulser cuvettes, and 1.5mL tubes at -20°C. Prewarm 2L LB+amp flasks and SOC @ 37°C. 
SECTION: Electroporation of STARR-seq Plasmid Library into Bacterial Cells
63. Add all 10μL of each gibson sample to a pre-cooled tube (2.5μL for controls). Keep on ice. 
SECTION: Electroporation of STARR-seq Plasmid Library into Bacterial Cells
64. Thaw MegaX DH10B cells on ice. Each tube contains ~120μL. 
SECTION: Electroporation of STARR-seq Plasmid Library into Bacterial Cells
65. Add 80μL of thawed cells to each 10μL gibson sample and 20μL to each 2.5μL control using pre-cooled tips. Keep on ice. 
SECTION: Electroporation of STARR-seq Plasmid Library into Bacterial Cells
66. Pipette SLOWLY up/down to mix with pre-cooled tips. Then add 20μL (max volume) to a GenePulser 0.1cm cuvette—may need to use 3-4 cuvettes for each sample, but only one for each control. Keep cuvettes on ice. 

Note: make sure the volume is touching both metal plates, otherwise it will arc (the best technique here is to pipette into the corner of the cuvette at an angle so the cells reach the bottom). To help, gently tap to the bottom.
SECTION: Electroporation of STARR-seq Plasmid Library into Bacterial Cells
67. Electroporate with “exponential decay” pulse type at 2kV, 200Ω, and 25μF - (Gap distance is 0.1cm for brown-capped cuvettes). 
SECTION: Electroporation of STARR-seq Plasmid Library into Bacterial Cells
68. Immediately add 1mL pre-warmed SOC media to the 20μL DNA/bacteria mixture in the cuvette. 
SECTION: Electroporation of STARR-seq Plasmid Library into Bacterial Cells
69. Combine like-reactions into a round bottom 14mL culture tube.
SECTION: Electroporation of STARR-seq Plasmid Library into Bacterial Cells
70. Recover at 37°C for 1 hour in shaker incubator. 
SECTION: Electroporation of STARR-seq Plasmid Library into Bacterial Cells
71. After recovery make a dilution series: 
  
SECTION: Electroporation of STARR-seq Plasmid Library into Bacterial Cells
72. Plate 100μL of each dilution onto a prewarmed LB+ampicillin(100μg/mL) plate. Incubate at 37°C for ~16hrs. 
SECTION: Electroporation of STARR-seq Plasmid Library into Bacterial Cells
73. With the remaining undiluted culture, add directly to 1L pre-warmed liquid LB+ ampicillin(100μg/mL) media. Shake at 37°C O/N 225 RPM. Do not do this for the controls! 

DO NOT FORGET TO ADD THE AMPICILLIN TO THE 1L CULTURE!!!
SECTION: Quantification of colony counts to estimate number of transformants
74. Count colonies on plates after overnight growth either manually or via automated means (imaging and particle counting with imageJ)

The equations below assume a plating of 100µL from a 1mL dilution preparation:
(.01 colony count) * 1000 = # colonies in 1mL of recovery media
(.001 colony count) * 10000 = # colonies in 1mL of recovery media
(.0001 colony count) * 100000 = # colonies in 1mL of recovery media
SECTION: Purification of Large Scale Plasmid Library Growth
75. Record OD600.
SECTION: Purification of Large Scale Plasmid Library Growth
76. Pellet 1L culture at 3400xg for 10min.
SECTION: Purification of Large Scale Plasmid Library Growth
77. Proceed with Zymo Plasmid GigaPrep Kit protocol according to manufacturer directions.
SECTION: Purification of Large Scale Plasmid Library Growth
78. Elute with 2mL ZymoPURE Elution Buffer. Nanodrop for purity and concentration. Store DNA at -20°C. 
SECTION: Low-Read Sequencing of Plasmid Library to Determine Insert Complexity
79. *Before proceeding to electroporation, it is important to determine whether the library has sufficient complexity. To do this, perform limited-cycle PCR to add illumina adapters and amplify the insert off the plasmid. Then submit the sample sequencing and target 15 million reads. Must be PE, but the cycle number is irrelevant as long as reads can be mapped. 

* To reduce rxn bias effect, perform two PCRs for each sample.

Prepare a mastermix: 
  
SECTION: Low-Read Sequencing of Plasmid Library to Determine Insert Complexity
80. Aliquot 50μL mix into PCR tubes. 
SECTION: Low-Read Sequencing of Plasmid Library to Determine Insert Complexity
81. Vortex and centrifuge, briefly. Run the following PCR reaction: 

98ºC, 30 sec
8 cycles of 
98ºC, 10 sec
68ºC, 30 sec
72ºC, 30 sec
Final extension 72ºC, 2 min
SECTION: Low-Read Sequencing of Plasmid Library to Determine Insert Complexity
82. Pool like-reactions and then purify using Zymo Clean and Concentrate - 5 kit – elute in 30μL elution buffer (warm buffer to 55°C or 37°C prior to elution and incubate on column for 5min).
SECTION: Low-Read Sequencing of Plasmid Library to Determine Insert Complexity
83. Determine concentration and size distribution via HS D5000 ScreenTape on the Agilent Tapestation. 

Concentration should be greater than 5nM but not much higher. Anything greater than 50nM is significantly overamplified.
SECTION: Low-Read Sequencing of Plasmid Library to Determine Insert Complexity
84. Submit samples for paired end sequencing. In the NAR paper, we requested 25M reads at PE150 on NovaSeq. 
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
85. *Each transfection reaction has a max capacity of 5 x106 cells and 10μL DNA. Do 20 reactions to reach 100x106 cells per sample. 

*To assess transfection efficiency, conduct an eGFP electroporation and no DNA electroporation (as unstained control). For this, only do one reaction of 5 million cells. The eGFP plasmid is ~2x the size of the ATAC-STARR library plasmids, so to match the required molarity, increase the concentration 2-fold. This control should be done for each individual cell type used in the experiment.

Prepare DNA samples: 
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
85.1. Add 110μg ATAC-STARR library DNA into a 1.5 mL Eppendorf tube in a max volume of 220μL [This equates to 5μg DNA per 5 million cells].
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
85.2. For GFP control, add 11μg of pcDNA3-eGFP plasmid (endotoxin-free) to a 1.5mL Eppendorf tube in a max volume of 11μL. [This equates to 10μg DNA per million cells]. 
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
85.3. For no DNA control, add 11μL water to a 1.5mL Eppendorf tube.
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
86. Prepare Suspension Cells: 
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
86.1. Day 1: Perform cell count (with hemocytometer)
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
86.2. Per sample, add 50 mL of warm recovery medium (without antibiotics) into four T-75 culture flasks. Incubate at 37°C until use. 
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
86.3. For each GFP or noDNA control reaction, add 10mL of media into a T-12.5 culture flasks. 
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
86.4. Transfer 121 million cells to as many 50 mL falcon tube as necessary and centrifuge at 300xg for 5 minutes. Aspirate supernatant. Wash and resuspend with 45mL of DPBS or PBS, combine pellets. Centrifuge for 300xg for 5 min. 
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
86.5. Set up the Neon Pipette Station:

a. Fill a fresh neon tube with 3 mL of pre-warmed Buffer E2. The electrode on the side of the tube should be     completely submerged in buffer. 
    
b. Insert tube into the pipette station until you hear a click. 
    
c. Replace neon tube between samples—only need to change between experimental and controls (can use the same     tube for water and GFP if doing water first). 
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
86.6. Resuspend cells in 2178μL Buffer R/T. (R for GM12878, GM11831, and SUDHL-6 and T for LCL8664). Gently pipette for a single cell suspension. (Do not leave cells resuspended in Buffer R/T for longer than 15-30 min).
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
87. Electroporation:
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
87.1. Add 1980μL cells to each ATAC-STARR library DNA and gently mix.
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
87.2. Add 99μL cells to GFP plasmid. Gently mix. 
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
87.3. Add 99μL cells to water. Gently mix. 
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
87.4. Electroporate 20 ATAC-STARR reactions and both control reactions (100μL at a time) using the following conditions: 

100μL Neon Tip, 1100V, 30ms, 2 pulses. 

If the sample arcs, eject into waste and grab a new tip—19 good reactions are better than 19 good + 1 very dead. 

Note: Each tip can be used 5 times. Reuse the tip only if doing an aliquot of the same sample. After use, save the tip and it can be regenerated to use for another application. Regenerated tips are somewhat less efficient and more prone to arcing than fresh tips, so do not use regenerated tips in this protocol. That said, these tips are quite expensive at ~$20 each, so keep that in mind when using. 
SECTION: Electroporate ATAC-STARR Libraries into Mammalian Cells
87.5. After each electroporation, remove the pipette from the station and expel contents into one of the prepared T-75 (or T-12.5 for control reactions) flasks from step 6 above. Repeat electroporation with remaining aliquots and expel 5 aliquots into each T-75 flask containing 50mL media—cell concentration will be ~500,000 cells/mL. Gently swirl flask after addition of each transfected aliquot. Place in incubator at 37°C for 24 hours. 
SECTION: Check transformation efficiency via flow cytometry
88. ***We will use PI as a viability dye; PI will stain dead cells***

After ~24 hours, for both GFP and No DNA transfections, prepare cells for flow:
SECTION: Check transformation efficiency via flow cytometry
88.1. Spin down cells in 15mL conical at 300xg, 5min. 
SECTION: Check transformation efficiency via flow cytometry
88.2. Remove supernatant and resuspend in 5mL 1x PBS or DPBS. 
SECTION: Check transformation efficiency via flow cytometry
88.3. Spin at 300xg, 5min. Remove Supernatant. 
SECTION: Check transformation efficiency via flow cytometry
88.4. Resuspend in 1000μL Flow Cytometry Buffer (1%BSA in 1xPBS) to achieve a cell density of ~1.25x106 cells/mL. 
SECTION: Check transformation efficiency via flow cytometry
88.5. Split each sample into two aliquots of 500μL each. Pass through the flow cytometry tube cap filter to obtain single-cell suspensions. 
SECTION: Check transformation efficiency via flow cytometry
88.6. Stain one from each with propidium iodine:

Make a 1:1 dilution of 1μg/μL PI solution with 1xPBS+1%BSA.

Add 1.25μL of diluted PI to the desired tubes. Vortex.
SECTION: Check transformation efficiency via flow cytometry
89. Analyze samples with 3-laser or 5-laser Flow Cytometer. Use no DNA as unstained compensation. No DNA+PI and GFP noPI as compensation controls. Analyze viable GFP+PI cells for GFP/no GFP. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
90. Exactly 24 hours after electroporation (counting from last transfection added to media), mix all four T-75 flasks—200mL should all fit in one. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
91. Take 10 µl sample from each flask and perform cell count/assess viability.
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
92. Divide into two 100mL volumes and process each separately. 

Note: one is for RNA harvest, the other is for isolated plasmid DNA harvest. Process the RNA aliquot first as it is the most time-sensitive.
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
93. Before processing each, spin down in 50mL conical tubes, 300xg 5min. Remove sup and wash with 25mL 1x PBS or 1x DPBS. Spin down cells at 300xg, 5min and remove supernatant. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94. RNA Extraction:

Note: Trizol and Chloroform steps should be done in a chemical fume hood. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.1. Add 5mL Trizol per sample (this equates to 1ml Trizol per 10e6 cells). Pipette up/down to homogenize. Note: can stop here and store at -20°C overnight.
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.2. Centrifuge 5 Invitrogen™ Phasemaker™ Tubes at 12,000xg for 30s. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.3. Aliquot five volumes of 1mL into Invitrogen™ Phasemaker™ Tubes. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.4. Incubate for 5 min at RT. Note: can stop here and store at -20°c overnight.
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.5. Add 0.2mL chloroform to each 1mL aliquot and mix vigorously by inversion. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.6. Incubate for 2-3min at RT. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.7. Spin at 12,000xg for 15min @4°C.
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.8. Transfer the aqueous layer to a 1.5mL Eppendorf tube—try to get ~600μL from each. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.9. Add 2μL Glycoblue co-precipitant (15mg/ml) to each. Do not combine like-reactions yet.
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.10. Add 0.5mL isopropanol to each aqueous phase.
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.11. Incubate at RT for 10min. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.12. Spin for 10min at 12,000xg @4°C.
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.13. Check for a white gel-like precipitate. If there, remove sup and continue with protocol. If no pellet spin again. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.14. Resuspend Centrifuge 5 Invitrogen™ Phasemaker™ Tubesat 12,000xg for 30s. pellet in 1mL 75% EtOH. RNA can be stored in this solution at -20°C for up to a year. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.15. Vortex briefly, and centrifuge 5min at 7500xg @4°C. Completely discard supernatant. It helps to use a P10 after briefly spinning.
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.16. Air-dry pellet for 5-10min. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.17. Combine by suspending all 5 pellets in 100μL nuclease-free water. Incubate @ 55°C for 10min.
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
94.18. Determine RNA yield with Nanodrop (expected yield: 2.5-1.5μg/μL, 260/280 > 2, 260/230 > 1.8). RNA can be stored at -80°C overnight. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
95. Plasmid DNA extraction: 

*Use ZymoPURE II midiprep to isolate plasmid DNA
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
95.1. Follow manufactures protocol for kit. Add 8mL P1 buffer directly to cell pellet. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
95.2. Elute in 50μL pre-warmed 10mM Tris pH 8.0. Incubate 5min on column. 
SECTION: Harvest RNA and plasmid DNA from ATAC-STARR transfected cells
95.3. Determine concentration and purity via nanodrop (expect ~300-400ng/μL). Store at -20°C. These samples will be amplified in a PCR reaction in the NGS PCR step.
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
96. *The magnetic beads are supplied at 5mg/mL and each mg can bind up to 5μg Poly(A)+ RNA. We will assume that Poly(A)+ RNA makes up about 5% (this is at the higher end because we are accounting for both mRNAs and the Reporter RNAs). Therefore, for 100μg Total RNA, we expect 5μg to be Poly(A)+ RNA. To capture all 5μg, we need 1mg of magnetic beads. Therefore, the ratio is 1μg Total RNA:10μg beads.

Determine the amount of beads to wash: 

  
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
97. Chill Wash Buffer B at 4°C.
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
98. Wash magnetic beads: 
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
98.1. Resuspend magnetic beads in the vial (vortex for 30s).







SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
98.2. Transfer the desired amount to a 1.5mL Eppendorf tube (use more than one tube if volume > 750μL). 
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
98.3. Add an equal volume of Binding Buffer and resuspend. 
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
98.4. Place tube on DynaMag-2 magnet and incubate for 1min. Remove supernatant. 
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
98.5. Remove tube from magnet and resuspend beads in the same volume of binding buffer used in step 98.3
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
99. Isolate Poly(A)+ RNA from Total RNA: 
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
99.1. Add 100μL binding buffer to each 100μL Total RNA sample.
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
99.2. Heat to 65°C for 2min to disrupt secondary structures. 
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
99.3. Immediately place on ice. Incubate 1min. 
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
99.4. Add the indicated amount of washed beads to add to the 200μL Total RNA solution. 

Note: Gently handle the beads for the remaining steps of the protocol. 
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
99.5. Mix well and incubate on a rotator for 5min at 4°C.  
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
99.6. Place tube on the magnet and incubate for ~30s. Remove supernatant. 
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
99.7. Remove tube from magnet and add the same volume used in step iv of Washing Buffer B. Mix by pipetting up/down gently. 
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
99.8. Rotate for 2-3min at 4°C. 
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
99.9. Incubate on magnet for ~30s and remove supernatant. 
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
99.10. Repeat wash for a total of 4 washes.
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
99.11. Add 82μL of 10mM Tris-HCl pH 7.5 to the beads. 
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
99.12. Gently mix. Incubate at 75°C for 2min. 
SECTION: Poly(A)+ RNA enrichment with oligo(dT)25 magnetic beads
99.13. Place on magnet and quickly transfer eluate to a 1.5mL tube. Determine concentration via nanodrop (should be ~500-250ng/μL). Note: Can stop here and store at -70°C.
SECTION: DNaseI digestion
100. *20 units are sufficient to treat ~100μg RNA

Per sample, add 10μL DNaseI Reaction buffer and 10μL DNase I (RNase-free)(20 units). 
SECTION: DNaseI digestion
101. Mix well and incubate @ 37°C for 10min. 
SECTION: DNaseI digestion
102. Purify Poly(A)+ RNA with Zymo RNA Clean & Concentrator – 25 kit. 

Volumes: 200μL RNA binding buffer, 300μL 100% EtOH

Elute in 60μL Nuclease-Free water. 
SECTION: DNaseI digestion
103. Nanodrop to determine concentration. 
SECTION: Reverse Transcription
104. For each 20μL reaction, the input maximum is 1μg Poly(A)+ RNA. We expect to have ~25μg, so scale up 25-fold. Do ten 50μL reactions. 

Anneal primer to template RNA:
SECTION: Reverse Transcription
104.1. Make MasterMix following the recipe below:
   
SECTION: Reverse Transcription
104.2. Aliquot 25μL into ten 0.2mL PCR tubes. 
SECTION: Reverse Transcription
104.3. Heat the RNA-primer mix at 65°C for 5min and then immediately incubate on ice for at least 1 min.
SECTION: Reverse Transcription
105. Prepare RT reaction mix: 
SECTION: Reverse Transcription
105.1. Vortex and briefly centrifuge the 5x PrimeScript Buffer
SECTION: Reverse Transcription
105.2. Make MasterMix following the recipe below: 
  
SECTION: Reverse Transcription
106. Add 25μL of the MasterMix to each of the 10 RNA-Primer reactions. Mix well. 
SECTION: Reverse Transcription
107. Incubate at 42°C for 60min.
SECTION: Reverse Transcription
108. To inactivate the reaction, heat at 70°C for 15min and then cool on ice. 
SECTION: RNaseA Treatment
109. Pool like reactions for a total volume of ~500μL
SECTION: RNaseA Treatment
110. Add 1μL RNAseA (10mg/mL) and incubate 37°C for 1hr.

Note: final concentration is ~20μg/mL in low salt conditions [KCl] = 75mM and [MgCl2] = 8mM. At low salt concentrations, RNase A cleaves single-stranded and double-stranded RNA as well the RNA strand in RNA-DNA hybrids.
SECTION: RNaseA Treatment
111. Purify cDNA using Zymo DNA Clean and Concentrate-25 kit:

a.Add 3.5mL Binding Buffer (7:1). Mix in a 15mL conical. Spin through column in 800μL increments. 
b.Wash with 200μL wash buffer twice. 
c.Elute in 28μL elution buffer.
SECTION: RNaseA Treatment
112. Nanodrop to determine yield and purity. Expected yield is ~10-50 ng/μL
SECTION: Next-Generation sequencing PCR
113. * To reduce rxn bias effect, perform two PCRs for each sample.

Prepare a mastermix: 
 
  
SECTION: Next-Generation sequencing PCR
114. Aliquot 39μL mix into PCR tubes and add 10μL of sample to each. 
SECTION: Next-Generation sequencing PCR
115. Add 1μL of the correct 50μM P7 Nextera primer (N7XX).
SECTION: Next-Generation sequencing PCR
116. Vortex and centrifuge, briefly. Run the following PCR reaction: 

98ºC, 30 sec
8 or 12 cycles of àplasmid DNA = 8 cycles, reporter RNA = 13 cycles
98ºC, 10 sec
68ºC, 30 sec
72ºC, 30 sec
Final extension 72ºC, 2 min
SECTION: Next-Generation sequencing PCR
117. Pool like-reactions and then purify cDNA using Zymo Clean and Concentrate -5 kit – elute in 30μL elution buffer (warm buffer to 55°C or 37°C prior to elution and incubate on column for 5min).
SECTION: Next-Generation sequencing PCR
118. Determine concentration and size distribution via HS D5000 ScreenTape on the Agilent Tapestation. Concentration should be greater than 5nM but not much higher. Anything greater than 50nM is significantly overamplified.

Note: you will see the template and initial amplification products in the Tapestation, these products are not capable of generating reads on the sequencer.
SECTION: Next-Generation sequencing PCR
119. Submit samples for paired end sequencing. In the NAR paper, we requested 50M reads for the DNA samples and 75M for the RNA samples at PE150 on NovaSeq.