TITLE: Loss of Function Mutagenesis Protocol
AUTHORS: Lilia Peyton, Ada McCarroll, Hanwen Zhang

[DESCRIPTION]
This protocols offers a description of the loss of function mutagenesis procedure for induced pluripotent stem cells.

[STEPS]
SECTION: iPSC Maintenance
1. Change media every other day – mTeSR Plus with 1ml primocin
SECTION: iPSC Maintenance
1.1. Always warm media to room temperature before use
SECTION: iPSC Maintenance
1.2. Aspirate using glass pipette attached to vacuum and replenish with mTeSR plus 
SECTION: Passaging
2. Passage every 4-6 days (whenever colonies are 70-80% confluent- depends on the density that you plate) 
SECTION: Passaging
2.1. Coat desired number and size of plates with matrigel/geltrex and incubate overnight or >4hr at 37C
SECTION: Passaging
2.2. On day of passage, cool the coated plates at room temperature for >1hr then aspirate matrigel/geltrex then add appropriate amount of media for well size 
SECTION: Passaging
2.3. Aspirate media from cells and wash with DBPS
SECTION: Passaging
2.4. Add appropriate amount of ReLeSR and aspirate within 1 minute 
SECTION: Passaging
2.5. Incubate at RT for 4-5 minutes 
SECTION: Passaging
2.6. Spray wells with media (1ml/well for 6w) with p1000 then hold the plate in one hand and use other hand to firmly tap the side of the plate to detach the cells 
SECTION: Passaging
2.7. Use p1000 to wash/collect the detached cells- gently pipet the cells 2-4 times, being careful not to break the aggregates into single cells and transfer to labeled 15ml tube 
SECTION: Passaging
2.8. Plate the cell aggregate mixture at the desired density onto the pre-coated wells containing media
Usually 1:10 - 1:20 or 1:4-1:5 depending on cell density 
SECTION: Passaging
2.9. Check that there is an appropriate number of cells under the microscope then place in the incubator at 37C; Move the plate in several quick, short, back-and-forth and side-to-side motions to evenly distribute the cells 
SECTION: Passaging
2.10. Do not disturb the plate for 24 hours 
SECTION: Thawing Cells
3. Prepare coated 6w plates with 1 well for each cryovial (same as step 1 above)
Can prepare 2 wells for each cryovial – depending on how confluent you want cells to be thawed
SECTION: Thawing Cells
4. On day of thawing, cool plates at RT >1hr before use but do not add media
SECTION: Thawing Cells
5. Label 15ml tubes for each cryovial that is being thawed
SECTION: Thawing Cells
6. Take cryovials out of liquid nitrogen
SECTION: Thawing Cells
7. Thaw cryovials in dry bath until only one ice crystal is left
SECTION: Thawing Cells
8. Use a glass pipette to remove cells from cryovial and transfer to labeled 15ml tube
SECTION: Thawing Cells
9. Slowly add 5ml mTeSR Plus dropwise into 15ml tube to avoid shocking the cells – rotate the tube while adding media to ensure even distribution
SECTION: Thawing Cells
10. Centrifuge 3 mins at 300g
SECTION: Thawing Cells
11. Aspirate supernatant – okay to leave a little film to avoid disturbing the pellet
SECTION: Thawing Cells
12. 10.   Aspirate coating matrix from prepared plate
SECTION: Thawing Cells
13. Use glass pipette to add 2ml mTeSR Plus with 5µM ROCK-Inhibitor and forcefully mix to break up pellet
SECTION: Thawing Cells
14. Add 2ml/well cell suspension to coated plate
SECTION: Thawing Cells
15. Incubate at 37C and change media with regular mTeSR Plus the next day/24 hours after to remove ROCK-I
SECTION: Freezing Cells
16. Determine which wells on 6w plate are ready to be cryopreserved
SECTION: Freezing Cells
17. Prepare labels and vials (2 vials per clone) – use label machine to create labels, ex. “g1b1c1A1”
SECTION: Freezing Cells
18. Put label on vial and scan barcode on bottom of vial in excel to connect it to the right clone 
SECTION: Freezing Cells
19. Aspirate media from wells and wash with DPBS
SECTION: Freezing Cells
20. Add 1ml ReLeSR per well and aspirate within 1 minute
SECTION: Freezing Cells
21. Incubate at RT for 4-5 minutes
SECTION: Freezing Cells
22. Spray wells with media (1ml/well for 6 well) then hold the plate in one hand and use other hand to firmly tap the side of the plate to detach cells
SECTION: Freezing Cells
23. Use P1000 to wash/collect the detached cells – gently pipet the cells 3-4 times, being careful not to break cell aggregates into single cells – and transfer  to labeled 15 ml tube
SECTION: Freezing Cells
24. Centrifuge for 3 min at 300g
SECTION: Freezing Cells
25. Gently aspirate the supernatant without disturbing the pellet
SECTION: Freezing Cells
26. Use 5ml glass pipette to resuspend pellet in 2 ml mFreSR
SECTION: Freezing Cells
27. 10.   Transfer 1ml of cell aggregate mixture into each labeled cryovial
SECTION: Freezing Cells
28. 11.   Transfer cryovials into isopropanol container then put it in -80C freezer for 48 hrs
SECTION: Freezing Cells
29. 12.   Transfer cryovials from isopropanol containers to boxes from LN2 on dry ice
SECTION: Freezing Cells
30. Need three people – one checks which box the vial should go in, one scans it into the excel spreadsheet, one physically moves vial from isopropanol container to box on dry ice; Transfer all vials at same time to ensure correct placement and ease for future shipment
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
31. Cell are ready to be plated for transfection when they are 70-80% confluent. Use 3-5 wells from a 6w plate depending on confluency 
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
32. Coat 2-24w plates 1 day before experiment – incubate at 37C overnight
Plate two different densities and determine which to use on day of transfection based on confluency and cell survival
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
33. On day of experiment, cool plates >1hr and set out appropriate amounts of media and accutase
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
34. Aspirate coating matrix and add 0.5ml media to each well – return plates to incubator until ready to use
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
35. Aspirate media and wash cells with DPBS
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
36. Add 1ml/well accutase and incubate at 37C for 5 min; if cells are not easily detaching incubate 7-10 mins 
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
37. While incubating, add 2ml media to 15ml tube
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
38. Following incubation, gently pipet cells twice and transfer to the prepared 15ml tube
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
39. Centrifuge 300g for 3 min
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
40. Aspirate supernatant – okay to leave a little film to not disturb pellet
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
41. Resuspend the pellet with a glass pipette in 4ml media – take 10µl for counting
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
42. Count cells using hemocytometer or countess machine
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
43. Calculate 2e5 and 1.5e5 cells per well 
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
44. Resuspend then aliquot with glass pipette for 25 wells into a new 50ml tube; repeat for both densities 
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
45. Centrifuge 300g for 3 min
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
46. Remove plates from incubator and label for each condition 
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
47. Aspirate supernatant – okay to leave a little film to not disturb pellet
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
48.  Resuspend cells in 12.5ml media
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
49.  Add 0.5ml cell suspension for each well in 24w plate
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
50. After every 6 wells, move the plate in several quick, short, back-and-forth and side-to-side motions to evenly distribute the cells
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
51. Incubate at 37C overnight
SECTION: Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
52. Repeat steps 17-21 for other density
SECTION: Transfection for iPSCs in 24w Plate
53. Before transfection, decide which density has better cell survival and density
SECTION: Transfection for iPSCs in 24w Plate
54. Change media for selected plate with antibiotics free mTeSR Plus with 5µM ROCK-I (700ul per well)
SECTION: Transfection for iPSCs in 24w Plate
55. Prepare working plasmid solution using endo-free TE buffer in hood: 
pEF-AncBE4max (from Brafman): 500ng/µl
pEF-BFP: 200ng/µl
pDT-sgRNA (with variant specific gRNA cloned): 300 ng/µl
SECTION: Transfection for iPSCs in 24w Plate
56. Prepare tubes for each sgRNA, one for DNA master mix, and one for Lipo master mix (26 tubes)
SECTION: Transfection for iPSCs in 24w Plate
57. Prepare DNA MM: 
625ul Opti-MEM
37.5ul AncBE4max
37.5ul BFP
SECTION: Transfection for iPSCs in 24w Plate
58. Aliquot 28ul of DNA MM into each sgRNA tube
SECTION: Transfection for iPSCs in 24w Plate
59. Add 1µl of each sgRNA to their corresponding tubes containing DNA MM
SECTION: Transfection for iPSCs in 24w Plate
60. Prepare Lipo Master mix: 
625ul Opti-MEM
82.5ul Lipofectamine STEM 
SECTION: Transfection for iPSCs in 24w Plate
61. Aliquot 28.3ul of LIPO MM to each sg RNA tube and mix well 
SECTION: Transfection for iPSCs in 24w Plate
62. Incubate at RT for 10 min
SECTION: Transfection for iPSCs in 24w Plate
63. Towards end of incubation, label prepared 24w plate with corresponding gene numbers 
SECTION: Transfection for iPSCs in 24w Plate
64. Add 50µl complex dropwise into the correct well and gently swirl plate to ensure even distribution
SECTION: Transfection for iPSCs in 24w Plate
65. Return plate to incubator
SECTION: Transfection for iPSCs in 24w Plate
66. 24 hours post transfection, refresh media with regular mTeSR Plus
SECTION: Transfection for iPSCs in 24w Plate
67.  48 hours post transfection, refresh media with regular mTeSR Plus
SECTION: Transfection for iPSCs in 24w Plate
68.  48 hours post transfection, coat 24 – 96w plates for sorting
SECTION: Transfection for iPSCs in 24w Plate
69. 72 hours post transfection, prepare for FACS
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
70. Check 24w plate with confocal microscope before preparing for FACS – determine that there is enough GFP+ cells to sort
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
71. Set out media and accutase to warm to RT – add CEPT components (1:10,000 chroman I, emricasan, and transISRIB; 1:1,000 polyamine supplement)  to media then filter before use
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
72. Cool 96w plates >1hr at RT then aspirate coating matrix and add 120µl media. Return plates to incubator for later use. 
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
73. Complete following procedure row by row (6 wells) for 4 batches of 6 wells. 
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
74. Wash with DPBS
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
75. Add 300µl accutase to each well
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
76. Incubate at 37C for 5 min; incubate longer 7-10 mins if needed 
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
77. During incubation, label 15ml tubes for each condition and add 1 ml of media to each tube 
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
78. Following incubation, gently pipet cells once and transfer to corresponding 15ml tubes 
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
79. Centrifuge 300g for 3 min
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
80. Aspirate supernatant – careful not to disturb pellet
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
81.  Resuspend pellet in 700µl media; 500ul if low cell confluence and 1ml if cell confluence is high
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
82. Filter cell suspension twice using two different 5ml corning round bottom tubes with blue strainer cap
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
83. Replace the blue cap with a filterless white cap from a sample collection tube to avoid contamination
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
84. Put samples on ice immediately
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
85. Sorting with FACS Aria – 1 double positive cell (BFP + GFP) into one well on 96w plate
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
86. After sorting, centrifuge the plates at 300g for 1 min to help cells attach
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
87. Return plates to incubator ASAP
SECTION: Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
88. Repeat until all 4 batches are done
SECTION: Care after Sorting *All media changes with regular mTeSR Plus to dilute CEPT cocktail)
89. 24hr post sorting: do not disturb cells
SECTION: Care after Sorting *All media changes with regular mTeSR Plus to dilute CEPT cocktail)
90. 48hr post sorting: use robot pipettor to add 50µl media
SECTION: Care after Sorting *All media changes with regular mTeSR Plus to dilute CEPT cocktail)
91. 72hr post sorting: use robot pipettor to change 50µl media
SECTION: Care after Sorting *All media changes with regular mTeSR Plus to dilute CEPT cocktail)
92. 96hr (Day 4) post sorting: use robot pipettor to change 120µl media
SECTION: Care after Sorting *All media changes with regular mTeSR Plus to dilute CEPT cocktail)
93. 144hr (Day 6) post sorting: aspirate 120µl, add 100µl media
SECTION: Care after Sorting *All media changes with regular mTeSR Plus to dilute CEPT cocktail)
94. Afterwards, use robot pipettor to change 100µl media every other day
SECTION: Care after Sorting *All media changes with regular mTeSR Plus to dilute CEPT cocktail)
95. It takes 10-14 days for a single cell to grow into a colony with appropriate size for picking  Approximately one week post sorting, determine which wells have colonies and start to mark/plan for colony picking
SECTION: Colony Picking
96. Day before experiment: Determine which plates you will be picking from based on size and colony morphology – mark 12 best colonies with microscope; Each colony marked plate will go onto one row on two identical 96w plates (see example below) – one plate for maintaining cells and one plate for DNA extraction
SECTION: Colony Picking
97. Day before experiment: Coat one row on 96w plate for each plate that is ready to be picked- normally 2-3 plates for the first round of picking depending on growth rate, then another 2 plates for the second round
Ex. 8 marked plates = one full 96 well plate 
SECTION: Colony Picking
98. On day of the experiment, cool plates >1hr RT then aspirate coating matrix and add 80µl mTeSR Plus with 5µM ROCK-I to each well (maintenance plates); an uncoated empty 96w plate copy is needed for each coated plate for DNA extraction (DNA plates). Label plates to correspond to the correct genes and indicate whether they are the "DNA plate" or the "maintenance plate" 
SECTION: Colony Picking
99. Pick colonies from one marked plate at a time following the below procedures 
SECTION: Colony Picking
100. Aspirate 50-70µl media from marked wells so around 70-100µl left
SECTION: Colony Picking
101. Set p200 pipettor at 50µl and scrape in marked area of well ~30 seconds then collect as many aggregates as possible in 50µl volume
SECTION: Colony Picking
102. Transfer the 50µl aggregates into a well on the maintenance plate, mix well by pipetting 3 times 
SECTION: Colony Picking
103. Withdraw 50ul cell suspension from maintenance plate and add to DNA plate
SECTION: Colony Picking
104. Repeat well by well until row is completed
SECTION: Colony Picking
105. Repeat process until maintenance and DNA plates have all intended genes
SECTION: Colony Picking
106. Centrifuge maintenance and DNA plates at 300g for 3 min
SECTION: Colony Picking
107. Return maintenance plate to incubator
SECTION: Colony Picking
108. Quickly aspirate media without disturbing the cells from the DNA plate
SECTION: Colony Picking
109. Plate can be stored at -20C temporarily, no more than an hour – while picking other maintenance and DNA plate
SECTION: Colony Picking
110. Add 16µl DNA quick extract to well on DNA plate and gently scrape/flush the well bottom to collect all cell aggregates – one well at a time, on ice
SECTION: Colony Picking
111. Transfer 16µl solution into a 96w PCR plate – keeping the same format as the DNA plate
SECTION: Colony Picking
112. Briefly spin PCR plate
SECTION: Colony Picking
113. Run DNA extraction protocol on thermos cycler (1st step in Sanger sequencing workflow)
SECTION: Colony Picking
114. Follow Sanger sequencing protocol to get results in 2-3 days
SECTION: Sanger Sequencing
115. DNA Extraction -> Amplification PCR -> SAP -> Sequence PCR -> Purification -> Sequencing 
SECTION: DNA Extraction (cell lysate in 16ul solution)
116. Run DNA extraction protocol on thermocycler: 
a. 65C for 15 min
b. 68C for 15 min
c. 95C for 10 min
d. 4C for ever 
SECTION: DNA Extraction (cell lysate in 16ul solution)
117. Go directly to PCR Amp step, do not pause here! 
SECTION: PCR Amplification
118. Prepare working primer from stock solution (100uM to 10uM) 
SECTION: PCR Amplification
119. Prepare master mix for each gene and for appropriate number of wells, accounting for 20% extra: 
AmpPCR mix 1x (ul) 
Water 5.5
10X PCR Amplification Buffer 1
10X PCR Enhancer 1
50mM MgSO4 0.3
10mM dNTP 0.2
Primer F 0.2
Primer R 0.2 
Polymerase 0.1
SECTION: PCR Amplification
120. Transfer prepared AmpPCR mixes into corresponding rows on a 96w PCR plate (8.5ul per well)
SECTION: PCR Amplification
121. Add 1.5ul quick extracted DNA to plates and mix 3 times 
SECTION: PCR Amplification
122. Seal plates and briefly centrifuge 2000g ~5 seconds
SECTION: PCR Amplification
123. Run PCR Amp protocol on thermocycler: 
a. 95C for 10 min
b. 95C for 30 sec
c. 53C for 1 min 30 sec
d. 72C for 1 min
e. Step b-d 40 times
f. 72C for 10 min
g. 4C for ever
SECTION: PCR Amplification
124. Directly to SAP step or can pause here; leave plate at 4C <18 hrs 
SECTION: SAP Reaction (5ul mix + 5ul PCR product= 10ul total)
125. Prepare master mix: 
SAP mix 1x(ul)
Water 3.9
10x SAP buffer 0.5
SAP 0.5
E. coli exonuclease I 0.1
SECTION: SAP Reaction (5ul mix + 5ul PCR product= 10ul total)
126. Pipette 5ul SAP mix into each well on a 96w plate
SECTION: SAP Reaction (5ul mix + 5ul PCR product= 10ul total)
127. Add 5ul PCR product to each well on SAP plate, pipetting 3 times to mix 
SECTION: SAP Reaction (5ul mix + 5ul PCR product= 10ul total)
128. Briefly centrifuge 2000g ~5 sec
SECTION: SAP Reaction (5ul mix + 5ul PCR product= 10ul total)
129. Run SAP protocol on thermocycler: 
a. 37C for 50 min
b. 95C for 15 min
c. 4C for ever 
SECTION: SAP Reaction (5ul mix + 5ul PCR product= 10ul total)
130. May pause here, leave the plate at 4C <18hr, or go directly to Seq PCR step 
SECTION: Seq PCR (5ul mix + 5ul SAP product = 10ul total)
131. Prepare master mix following table above for each gene - only use either F primer or R primer, do not use both! Be aware of primer records, some genes require more of less big dye 

Seq PCR mix 1x (ul)
Water 2 
Big dye 2
Primer F or R

SECTION: Seq PCR (5ul mix + 5ul SAP product = 10ul total)
132. Pipette 5ul mix into a 96w PCR plate, being careful to be consistent with gene placement
SECTION: Seq PCR (5ul mix + 5ul SAP product = 10ul total)
133. Add 5ul SAP product, pipetting 3 times to mix
SECTION: Seq PCR (5ul mix + 5ul SAP product = 10ul total)
134. Seal the plate and briefly centrifuge 2000g ~5 sec
SECTION: Seq PCR (5ul mix + 5ul SAP product = 10ul total)
135. Run Seq PCR protocol on thermocycler: 
a. 96C for 1m in
b. 96C for 10 sec
c. 55C for 5 sec
d. 60C for 4 min
e. Step b-d for 25 times
f. 4C for ever 
SECTION: Seq PCR (5ul mix + 5ul SAP product = 10ul total)
136. May pause here, leave the plate at 4C <18 hrs or go directly to purification step 
SECTION: Purification
137. Prepare fresh 75% isopropanol
SECTION: Purification
138. Add 40µl 75% isopropanol into each well, pipetting 3 times to mix
SECTION: Purification
139. Gently attach a sealing film and incubate at RT for 20 min
SECTION: Purification
140. Centrifuge plate at 2700g for 30 min
SECTION: Purification
141. Gently invert the plate onto paper towel
SECTION: Purification
142. Add 150µl 75% isopropanol in each well, do not mix! 
SECTION: Purification
143. Reseal the plate and centrifuge at 2700g for 12 min
SECTION: Purification
144. Gently invert the plate onto a paper towel
SECTION: Purification
145. Keep the plate inverted and transfer to a new paper towel and centrifuge at 200g for 1 min to remove residual isopropanol
SECTION: Purification
146. 11.   Directly go to next step
SECTION: Prepare for Loading
147. Add 16µl HiDi formamide to each well, pipetting 3 times to mix 
SECTION: Prepare for Loading
148. Seal the plate and centrifuge at 2000g for ~5 sec
SECTION: Prepare for Loading
149. Run the forma protocol on thermocycler: 
a. 95C for 3 min
b. 4C for 2 min
c. 4C for ever 
SECTION: Prepare for Loading
150. Centrifuge at 2000g for 1 min
SECTION: Prepare for Loading
151. Ready to load
SECTION: Setup 3730 for Sequencing
152. Start 3730 software 
SECTION: Setup 3730 for Sequencing
153. Select instrument status
SECTION: Setup 3730 for Sequencing
154. Connect the polymer bottle – set waste bottle and cap on white tray
SECTION: Setup 3730 for Sequencing
155. Run bubble remove wizard twice (check for bubbles) then fill array
SECTION: Setup 3730 for Sequencing
156. In plate manager – import or build a new template for today’s run 
SECTION: Setup 3730 for Sequencing
157. Go to run scheduler – search the plate ID and add plate in the queue
SECTION: Setup 3730 for Sequencing
158. Make sure to remove any sealing film on PCR plate (will damage array if not)
SECTION: Setup 3730 for Sequencing
159. Put the PCR plate in the black bottom, add a clean grey septa on it, then attach the white top – make sure it clicks closed and have angled corner of black bottom on top right of PCR plate, A12 on PCR plate goes in angled corner to have correct orientation
SECTION: Setup 3730 for Sequencing
160. 10.   Put the PCR plate sandwich into the “In stack” drawer – make sure to close the door properly
SECTION: Setup 3730 for Sequencing
161. 11.   When the system detects plates in the “In stack”, click “play” button – top left in the software – then click “yes” to confirm and start the program
SECTION: Setup 3730 for Sequencing
162. 12.   Takes ~2 hours to run a full 96w plate
SECTION: Setup 3730 for Sequencing
163. 13.   After run, copy data to a flashdrive 
SECTION: Setup 3730 for Sequencing
164. 14.   Exit the software, shutdown the computer, and return the polymer to 4C
SECTION: Setup 3730 for Sequencing
165. Analyze data on computer with SeqScape 
SECTION: Setup 3730 for Sequencing
166. 18.   Check each sample for expected editing and record – use to decide which wells to expand
SECTION: Expand Selected Clones
167. 
SECTION: Expand Selected Clones
168. After viewing Sanger sequencing results, label 4 wells to keep on 96w plate – two to expand and two as backups; prioritize homozygous clones and clones with the best morphology 
SECTION: Expand Selected Clones
169. 24hr post picking, add 50µl regular mTeSR Plus into selected wells
SECTION: Expand Selected Clones
170. 48hr post picking, change 80µl media for selected wells
SECTION: Expand Selected Clones
171. Change 100µl media every other day
SECTION: Expand Selected Clones
172. Once colonies have grown to proper size (typically 4-6 days), expand from 96w to 6w
SECTION: Expand Selected Clones
173. Coat one well on 6w plate for each well on 96w plate that is ready to be expanded – incubate >4hr
SECTION: Expand Selected Clones
174. Cool 6w plates >1hr at RT and label for each well being expanded – cell line, gene #, clone, gene name, passage #
SECTION: Expand Selected Clones
175. Aspirate coating matrix and add 2ml media to each well
SECTION: Expand Selected Clones
176. Then aspirate media from wells ready to be expanded with p200
SECTION: Expand Selected Clones
177. Add 100µl ReLeSR to wells and immediately aspirate – add and aspirate from wells one by one
SECTION: Expand Selected Clones
178. Incubate for 3 min at RT
SECTION: Expand Selected Clones
179.  Add 100µl media to each well
SECTION: Expand Selected Clones
180.  Scrape colonies one by one using p200 – scape entire area
SECTION: Expand Selected Clones
181. Aspirate full volume from well and add to corresponding well on the 6w plate – move back and forth, right and left to gently disperse cells
SECTION: Expand Selected Clones
182. Passage cells once to have 2 wells on 6w plates with appropriate confluence for each clone – one well is needed for cryopreservation and one well for RNA isolation
SECTION: RNA isolation
183. Add 800ul buffer RLT Plus to expanded 6w plates with 70-80% confluence
SECTION: RNA isolation
184. Pipet 30-60 seconds, washing the well until the solution is homogenized and clear
SECTION: RNA isolation
185. Add 350ul each to two locking lid 1.5 ml microcentrifuge tubes 
SECTION: RNA isolation
186. Store in -80C for temporary storage. Within a week, isolate RNA using the RNeasy Plus Mini Kit 
SECTION: RNA isolation
187. Transfer cell lysate to QIA shredder and centrifuge 2 min at max speed 
SECTION: RNA isolation
188. Transfer liquid to gDNA elimination column and centrifuge 15 sec at 11,000 rpm
Discard flow through and keep spin column and tube
SECTION: RNA isolation
189. Add 350ul 70% ethanol to lysate and mix until homogenized 
SECTION: RNA isolation
190. Transfer to RNA easy spin column and centrifuge 15 sec at 11,000 rpm
Discard flow through and keep spin column and tube
SECTION: RNA isolation
191. Add 700ul buffer RW1 to RNA easy spin column and centrifuge for 1 min at full speed
Discard flow through and keep spin column and tube
SECTION: RNA isolation
192. Add 500ul buffer RPE to RNA easy spin column and centrifuge 15 sec at 11,000 rpm
Discard flow through and keep spin column and tube
SECTION: RNA isolation
193. Repeat previous step, centrifuge for 2 min rather than 15 sec
SECTION: RNA isolation
194. Transfer RNA easy spin column to collection tube and centrifuge 1 min at full speed
SECTION: RNA isolation
195. Transfer RNA easy spin column in labeled 1.5l tube and add 35ul RNAse free water directly to the membrane 
SECTION: RNA isolation
196. Centrifuge for 1 min at 11,000 rpm
SECTION: RNA isolation
197. Discard RNA easy spin column and place 1.5ml tube directly on ice
SECTION: RNA isolation
198. Measure the concentrations of each sample using the NanoDrop
SECTION: RNA isolation
199. Aliquot 20-25ul into new labeled 1.5ml tubes and wrap well with parafilm to ship to Novogene
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
200. Fixation 
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
200.1. Coat coverslips the day before in 4w dish
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
200.2. Start from iPSCs culture on 96w plate- plate on 12mm 1.5 image-grade glass
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
200.3. Prepare 4% PFA solution in 1xPBS (16% stock in PBS, 400ul per well)
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
200.4. Aspirate old media and add 4% PFA into each well 
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
200.5. Incubate at RT for 12-15 min
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
200.6. Aspirate PFA, wash cells 3 times with PBS
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
200.7. After third wash, add 1ml PBS + 0.03% sodium azide to each well 
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
200.8. Wrap the 4w dish using parafilm and store the plate in 4C; you can pause her for a few days
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
201. Permeabilization
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
201.1. Prepare blocking buffer (3% BSA in 0.1% PBST)
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
201.2. Prepare 1% triton X-100 in PBS
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
201.3. Add 500ul PBST to each well and incubate at RT for 15 min
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
202. Blocking
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
202.1. Aspirate PBST from permeabilization step
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
202.2. Add 1ml blocking buffer to each well (3% BSA in 0.1% PBST (0.1% triton in 1x PBS))
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
202.3. Incubate RT for 1 hr; can leave in 4C overnight 
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
203. Primary Antibody 
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
203.1. Following vendors instructions, prepare antibodies in blocking buffer; can use 1ug/ml to start if no instructions are available 
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
203.2. Aspirate blocking buffer
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
203.3. Add 350ul primary antibodies in blocking buffer to each well and incubate RT for 1.5 hr; can leave overnight if necessary but increase the volume of solution 
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
204. Wash
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
204.1. Aspirate primary antibody
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
204.2. Add 1ml PBS or 0.1% PBST to each well - 3 times, incubating for 5 min at RT each time
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
205. Secondary Antibody 
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
205.1. Prepare 1:1000 dilution in blocking buffer; make sure to se different hosts and colors and match the primary antibody
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
205.2. Aspirate wash
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
205.3. Add 350ul secondary antibody in blocking buffer to each well and incubate at RT for 1 hr. It is light sensitive at this point so be sure to incubate in the dark (cover in foil, keep in a drawer or cabinet)
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
206. Wash
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
206.1. Repeat same washs step as above but incubate in the dark
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
207. DAPI
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
207.1. Ready to use solution in 4C fridge (0.5ug/ml in PBS)
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
207.2. Aspirate wash
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
207.3. Add 400ul DAPI solution to each well and incubate at RT for 10 min
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
208. Wash
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
208.1. Aspirate DAPI
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
208.2. Briefly wash each well with 1ml PBS
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
208.3. Thaw diamond anti-fade solution 
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
209. Mount
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
209.1. Add one drop of diamond anti-fade solution to slide
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
209.2. Aspirate PBS and use tweezers to grab and invert the coverslip to make sure the cells contact the anti-fade reagent 
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
209.3. Slides can be stored in the dark, > overnight to let the anti-fade dry
SECTION: Immunohistochemistry: Staining for Quality Control of iPSCs
209.4. Ready for imaging