Page Comparison

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•  Thaw and stage capture and lysis materials
•  Thaw one PIP tube per sample and RNase Inhibitor on ice
•  Stage at RT Partitioning Reagent, Chemical Lysis Buffer 3
•  Stage Combination 1.5 ml/0.5 ml tube stand, P200 and P1000 wide and standard
•  Stage one 0.5 ml tube per sample
•  Warm Cell Suspension Buffer to 37 C and cell suspension if frozen for 60-90 seconds
•  decontaminate cell vial with 70% isopropyl alcohol
•  thaw remaining ice at RT 30-60 sec
•  Use a wide bore P1000 to gently mix and transfer to 15 ml conical tube
•  Slowly add 9 ml warmed thawing media (>30s)
•  Centrifuge cells at 200 xg for 5 minutes to pellet
•  Aspirate as much supernatant as possible without disturbing pellet
•  Add 1 ml prewarmed Cell Suspension Buffer, gently mix with wide bore P1000
•  Place remaining Cell Suspension Buffer on ice
•  Centrifuge at 200 x g for 3 min
•  Aspirate as much supernatant as possible
•  Use standard bore P100 to add 200-400 ul cold Cell Suspension Buffer and gently mix to resuspend 10-15 times
•  Measure cell count
•  Use wide bore P1000 to prepare 1,250 live cells/ul in Cell Suspension Buffer
•  Measure cell count, repeat 9 and 10 until cell count reached
•  place on ice

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•  Centrifuge Thawed PIP tubes for ~5 seconds and return to ice
•  Preheat Dry Bath with 0.5 ml block to 25 C (lid +5C or 30 C)
•  Use P200 wide bore to gently mix cell suspension 10x
•  Add 4 ul cell suspension directly into PIPs
•  Add 1-2 ul RNase Inhibitor directly into PIPs slowly
•  Mix PIP mix with standard bore low retention P200 at 25 ul (avoid bubbles) 10x
•  Add 280 ul Partitioning Reagent down the side of tube
•  Cap tubes and place in the yellow rotating vortex adapter in horizontal config
•  vortex 3000 rpm 15 sec
•  rotate adapter to vertical config and vortex 3000 rpm for 2 minutes
•  Place PIP tubes in 0.5 ml side of stand. Let stand 30 s
•  Place a P200 set at 115 ul toward bottom of tube and wait 5 s then slowly aspirate
•  Repeat above: P200 at bottom, 5 s, slowly aspirate
•  Check Chemical Lysis Buffer 3 for crystals, if present warm w dry bath, vortex 10s, centrifuge
•  Add 40 ul CLB 3 to each 0.5 ml tube with low retention P200
•  Add 120 ul Partitioning Reagent to each 0.5 ml tube
•  Vortex 0.5 ml tube 10s, then immediately pipette 160 ul to PIP
•  Mix PIPs by inversion >10x
•  Insert PIP tubes into dry bath and select skip and yes to run:

HOLD POINT IN DRY BATH UP TO 96 HOURS

mRNA isolation

•  Thaw and stage materials
•  Thaw at RT Breaking Buffer for >20 m, DePartitioning Reagent
•  On ice Washing Buffer, RT Additive Mix V, one tube TSO per 2 samples
•  Stage and label per sample 1.5 ml Safe Locks, 1.5/0.5 ml stand, red PIPseq guide rack
•  Aliquot 1 ml Washing Buffer into 1.5 ml tubes and place in ice
•  0.2 ml 8 tube strips and lids
•  Remove PIPtubes and place in blue stand, allow to settle for 30s if needed
•  Place P200 at bottom of tube slowly, wait 5 s, aspirate ~130ul slowly, wipe tip along side slowly
•  Add 200 ul Breaking Buffer to each PIP along side
•  Add 40 ul DePartitioning Reagent to each PIP along side
•  Invert to break emulsion 10-20x
•  Centrifuge for 10s
•  Ensure distinct interface between pink bottom and cloudy top
•  if not, add 4 ul DePartitioning and repeat inversion and centrifuge
•  Remove ALL pink and red layers with P200
•  Centrifuge 10 s
•  Remove ALL remaining pink with a P20, place 0.5 ml tubes on ice
•  Use P200 to slowly transfer 180 ul PIPs to chilled Wash in 1.5 ml tubes
•  Briefly centrifuge 0.5 ml tubes
•  Repeat transfer of another 180 ul to 1.5 ml tubes
•  Place tubes in blue stand and vortex horizontally for 3 s
•  Centrifuge 1 m, turn off for gradual slow
•  Gently Place tubes back in rack
•  Aspirate slowly and discard supernatant to the 4 or L mark on stand
•  Wash2
•  Add 1 ml Washing Buffer, vortex in stand horizontally 3 sec, centrifuge 1 m
•  Gently place in rack, Aspirate slowly and discard supernatant to the 4 or L mark on stand
•  Wash3
•  Add 1 ml Washing Buffer, vortex in stand horizontally 3 sec, centrifuge 1 m
•  Gently place in rack, Aspirate slowly and discard supernatant to the 4 or L mark on stand
•  Wash4
•  Add 1 ml Washing Buffer, vortex in stand horizontally 3 sec, centrifuge 1 m
•  Gently place in rack, Aspirate slowly and discard supernatant to the 4 or L mark on stand
•  Move ~100 ul (ALL) into 0.2 ml tube (PCR strip) cap
•  Spin 5 s at 2000 xg and place in red rack
•  Let settle >1 minute
•  Reduce Volume to guide wire, then place on ice

cDNA synthesis

•  Prep Reagents
•  Thaw RT Enzyme Mix on ice
•  Prepare 1:1 Washing Buffer, 600 ul per sample, keep on ice
•  stage ultra pure water
•  Prepare RT Master Mix:

•  Mix well by pipetting, centrifuge briefly
•  Add 39 ul RT MM to each 0.2 ml well, mix well by pulse vortexing
•  Centrifuge <1 s
•  Thermocycle with lid at 105C:

HOLD POINT 4 C in Thermocycler overnight

•  Briefly centrifuge 0.2 ml tubes, then Add 120 ul 0.5X Wash Buffer, seal with new lid
•  Vortex mix for 5 s
•  Centrifuge 5 s, power off centrifuge to slow, and return to red rack
•  Tap guide rack 3 x on bench
•  Let settle 1 minute
•  Aspirate and discard 150 ul
•  Wash2
•  Add 150 ul 0.5 X Washing Buffer, vortex mix 5 s, centrifuge 5s
•  Tap 3 x, let settle 1 minute
•  Aspirate and discard 150 ul
•  Wash3
•  Add 150 ul 0.5 X Washing Buffer, vortex mix 5 s, centrifuge 5s
•  Tap 3 x, let settle 1 minute
•  Aspirate and discard 150 ul
•  Wash3
•  Add 150 ul 0.5 X Washing Buffer, vortex mix 5 s, centrifuge 5s
•  Tap 3 x, let settle 1 minute
•  Aspirate and discard 150 ul