TITLE: Dip-C (Part 2: Whole-genome Amplification with Nextera)
AUTHORS: Longzhi Tan

[STEPS]
?. [Oligos]
Carrier ssDNA (same as in LIANTI and META):TCAGGTTTTCCTGAAPurification: standard desaltingDissolve in 0.1 X TE (made from  ) to a final concentration of .Store at .
-20 °C
?. [Oligos]
Nextera i7 Index Primers:701: CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG702: CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGG703: CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGG704: CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGG705: CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGG706: CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGG707: CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGG708: CAAGCAGAAGACGGCATACGAGATCCTCTCTGGTCTCGTGGGCTCGG709: CAAGCAGAAGACGGCATACGAGATAGCGTAGCGTCTCGTGGGCTCGG710: CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGG711: CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGG712: CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGGand the following if > 96 cells need to be sequenced at the same time (e.g. NovaSeq):714: CAAGCAGAAGACGGCATACGAGATTCATGAGCGTCTCGTGGGCTCGG715: CAAGCAGAAGACGGCATACGAGATCCTGAGATGTCTCGTGGGCTCGG716: CAAGCAGAAGACGGCATACGAGATTAGCGAGTGTCTCGTGGGCTCGG718: CAAGCAGAAGACGGCATACGAGATGTAGCTCCGTCTCGTGGGCTCGG719: CAAGCAGAAGACGGCATACGAGATTACTACGCGTCTCGTGGGCTCGG720: CAAGCAGAAGACGGCATACGAGATAGGCTCCGGTCTCGTGGGCTCGG721: CAAGCAGAAGACGGCATACGAGATGCAGCGTAGTCTCGTGGGCTCGG722: CAAGCAGAAGACGGCATACGAGATCTGCGCATGTCTCGTGGGCTCGG723: CAAGCAGAAGACGGCATACGAGATGAGCGCTAGTCTCGTGGGCTCGG724: CAAGCAGAAGACGGCATACGAGATCGCTCAGTGTCTCGTGGGCTCGG726: CAAGCAGAAGACGGCATACGAGATGTCTTAGGGTCTCGTGGGCTCGG727: CAAGCAGAAGACGGCATACGAGATACTGATCGGTCTCGTGGGCTCGGPurification: standard desaltingDissolve in 0.1 X TE to a final concentration of .Dilute with 0.1 X TE to  in PCR tubes.
?. [Oligos]
Nextera i5 Index Primers:501: AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTC502: AATGATACGGCGACCACCGAGATCTACACCTCTCTATTCGTCGGCAGCGTC503: AATGATACGGCGACCACCGAGATCTACACTATCCTCTTCGTCGGCAGCGTC504: AATGATACGGCGACCACCGAGATCTACACAGAGTAGATCGTCGGCAGCGTC505: AATGATACGGCGACCACCGAGATCTACACGTAAGGAGTCGTCGGCAGCGTC506: AATGATACGGCGACCACCGAGATCTACACACTGCATATCGTCGGCAGCGTC507: AATGATACGGCGACCACCGAGATCTACACAAGGAGTATCGTCGGCAGCGTC508: AATGATACGGCGACCACCGAGATCTACACCTAAGCCTTCGTCGGCAGCGTCand the following if > 96 cells need to be sequenced at the same time (e.g. NovaSeq):510: AATGATACGGCGACCACCGAGATCTACACCGTCTAATTCGTCGGCAGCGTC511: AATGATACGGCGACCACCGAGATCTACACTCTCTCCGTCGTCGGCAGCGTC513: AATGATACGGCGACCACCGAGATCTACACTCGACTAGTCGTCGGCAGCGTC515: AATGATACGGCGACCACCGAGATCTACACTTCTAGCTTCGTCGGCAGCGTC516: AATGATACGGCGACCACCGAGATCTACACCCTAGAGTTCGTCGGCAGCGTC517: AATGATACGGCGACCACCGAGATCTACACGCGTAAGATCGTCGGCAGCGTC518: AATGATACGGCGACCACCGAGATCTACACCTATTAAGTCGTCGGCAGCGTC520: AATGATACGGCGACCACCGAGATCTACACAAGGCTATTCGTCGGCAGCGTCPurification: standard desaltingDissolve in 0.1 X TE to a final concentration of .Dilute with 0.1 X TE to  in PCR tubes.
?. [Reagents]
Prepare 60 mg/mL Qiagen Protease:Add  water to one vial of  .Vortex to mix.Filter to sterilize.Store at .
2.78 mL
4 °C
?. [Reagents]
Prepare Lysis Buffer ( per cell; recipe below for , or 4 96-well plates): (final: ) (final: ) (final: ) (aliquoted and stored at ; final: ) (final: ) 100 uM Carrier ssDNA (final: ) waterStore at  if needed.
2 µl
1 mL
20 µl
4 µl
15 µl
25 µl
-20 °C
2 µl
5 µl
929 µl
-20 °C
?. [Reagents]
Prepare Transposition Buffer ( per cell; recipe below for , or 1 96-well plate): (final: ) (final: ) (final: ) waterStore at  if needed.
8 µl
1 mL
12.5 µl
6.25 µl
200 µl
781.25 µl
-20 °C
?. [Lysis (Skip if Performing Positive Control)]
Prepare Dip-C Lysis Buffer ( per cell; recipe below for 4 96-well plate + 25%): Lysis Buffer 60 mg/mL Qiagen Protease (final: )Aliquot to  in 12-strip tubes.
2 µl
960 µl
0.24 µl
78 µl
?. [Lysis (Skip if Performing Positive Control)]
Add  Dip-C Lysis Buffer per well to a .
2 µl
?. [Lysis (Skip if Performing Positive Control)]
Seal the plate with  and .
?. [Lysis (Skip if Performing Positive Control)]
Centrifuge at   briefly.
Centrifuge: 1000 34
?. [Lysis (Skip if Performing Positive Control)]
Flow sort one cell per well (see Part 1 for details).
?. [Lysis (Skip if Performing Positive Control)]
Lyse the cells by running (set lid temperature to  to avoid evaporation;  volume; total: ~; minimize evaporation by sealing tight and closing the PCR machine lid tight): for  for  forever
75 °C
2 µl
50 °C
70 °C
4 °C
?. [Lysis (Skip if Performing Positive Control)]
Store at  if needed (stable for a few months). For longer storage at , sort cells into dry (empty) wells.
-80 °C
-80 °C
?. [Positive Control (Optional)]
Prepare 100 pg/uL gDNA in a  (recipe below for NEB HeLa gDNA; can be made from any human or mouse gDNA): water (final: )
1 mL
1 µl
?. [Positive Control (Optional)]
Prepare Positive Control Solution in a  ( per positive control; recipe below for ): water 100 pg/uL gDNA (final: )
2 µl
20 µl
19 µl
1 µl
?. [Positive Control (Optional)]
Add  Positive Control Solution per positive control well.
2 µl
?. [Reagents]
Prepare Transposome Removal Buffer ( per cell; recipe below for , or 4 96-well plates): (final: ) (final: ) (final: ; for ease of pipetting) waterStore at  if needed.
2 µl
1 mL
60 µl
90 µl
1 µl
849 µl
-20 °C
?. [Lysis (Skip if Performing Positive Control)]
Centrifuge at  .
Centrifuge: 1000 34, 1 min
?. [Transposition]
Make Transposition Mix ( per cell; recipe below for 96-well plate + 10%): Transposition Buffer~ 125 nM Homemade Nextera Transposome or Aliquot to  in 12-strip tubes.
8 µl
844.8 µl
1.6 µl
69 µl
?. [Transposition]
Add  Transposition Mix per well, avoiding touching the liquid.
8 µl
?. [Transposition]
Vortex and spin down.
?. [Transposition]
Transpose the genome by running ( volume; total: ~): for  forever
10 µl
55 °C
4 °C
?. [Stop]
Prepare Stop Mix ( per cell; recipe below for 96-well plate + 25%): Transposome Removal Buffer 60 mg/mL Qiagen Protease (final: )Aliquot to  in 12-strip tubes.
2 µl
240 µl
0.4 µl
19 µl
?. [Stop]
Stop transposition by running ( volume; total: ~): for  for  forever
12 µl
50 °C
70 °C
4 °C
?. [Amplification]
Make PCR Mix (~ per cell; recipe below for 96-well plate + 10%): Q5 Reaction Buffer (included with ) Q5 High GC Enhancer (included with )Aliquot to  in 12-strip tubes.
11 µl
528 µl
528 µl
63.36 µl
6.336 µl
26.4 µl
26.4 µl
97 µl
?. [Stop]
Add  Stop Mix per tube, avoiding touching the liquid.
2 µl
?. [Stop]
Vortex and spin down.
?. [Amplification]
Add (per tube; avoid touching the liquid): PCR Mix 12.5 uM Nextera i5 Primer (final: ) 12.5 uM Nextera i7 Primer (final: )
11 µl
1 µl
1 µl
?. [Amplification]
Amplify by running ( volume; total: ~): for  (to allow the lid to pre-heat) for  for 14 cycles of  for ,  for ,  for  for  forever
25 µl
4 °C
72 °C
98 °C
98 °C
62 °C
72 °C
72 °C
4 °C
?. [Amplification]
Store at  if needed.
-20 °C
?. [Purification]
Pool cells as desired and purify with  and  DNA Binding Buffer per cell (a 1:5 ratio; extra buffer can be purchased as ). Elute in  per cell.For a 96-well plate, pooling can be done with a multi-channel pipette into a total of   Binding Buffer. Use 4-6 columns per plate and elute into a total of .
125 µl
4 µl
12 mL
400 µl
?. [Purification]
Measure concentration with .
?. [Purification]
Measure size distribution with .
?. [Purification]
Remove short fragments with 0.7 X . Elute into .
?. [Purification]
Measure concentration with .
?. [Purification]
Measure size distribution with .