Page Comparison

Cell Preparation

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• 36 total samples to be processed in 6 batches

  • Batch1: 2 sample batch

  • Batch2: 2 sample batch

  • Batch3: 16 samples

  • Batch4: 16 samples

1Cell Preparation

•  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

...

•  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

•  Stage Reagents
•  Thaw on ice 4X PCR MM and WTA Primer
•  Stage two 1.5 ml tube and ultra pure water
•  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
•  Wash4
•  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
•  Reduce Volume to rack guidewire and place on ice

cDNA Amplification

•  Pipette mix WTA Primer and centrifuge briefly
•  Make 1X WTA Primer (3 ul WTA and 27 ul H2O for the 2 and 2) or (6ul WTA and 54 ul H2O for 8 )
•  Make PCR MM using pipette mixing for reagents

•  Add 21 ul MM into each PCR tube
•  Pulse vortex in black stand and quick spin down
•  Run on Thermocycler with 105 C lid:

HOLD POINT 4 C in Thermocycler overnight

Isolate cDNA from PIPs in Post PCR area

•  Stage QC reagents on ice
•  4X PCR MM
•  WTA Primer (do not dilute)
•  Washing Buffer
•  Stage Reagents at RT
•  CE Buffer, low EDTA TE, ultra pure water
•  Illumina Purification Beads (limit light exposure) >20 minutes
•  0.2 ml tubes or strip tubes and lids
•  Magnet rack
•  Add 40 ul CE Buffer to each WTA rxn and seal with new lids
•  Pulse vortex and spin down
•  Place in guide rack, tap on bench, and wait > 1 minute for PIPs to settle below guide
•  Transfer 60 ul supernatant into a NEW labeled 0.2 ml tube
•  Add another 60 ul CE Buffer to the original tubes
•  Pulse vortex, spin down 5s, load back into guide rack
•  Tap on bench and let settle >1 minute
•  Transfer 60 ul supernatant into tubes containing supernatant
•  Briefly centrifuge supernatant tubes.
•  If PIPs are present, transfer supernatant to new tubes
•  Save remaining PIP pellet at -80C as backup

MagBead Purification

•  Prepare at least 400 ul of fresh 85% Ethanol per rxn (340 ul EtOH and 60 ul ultra pure water)
•  Resuspend Illumina Purification beads via vortex <30s
•  Check Volumes of samples
•  Add 0.8x beads to each sample (96 ul beads for 120 ul sample or sampleX0.8 of beads)
•  Vortex until thoroughly mixed then pulse centrifuge to bring liquid to bottom
•  Incubate 5 min at RT
•  Place tubes in magnetic stand for 5 minutes or until liquid is clear
•  Remove supernatant <216 ul
•  Add 200 ul 85% EtOH, incubate 30 s, and discard
•  Add 200 ul 85% EtOH, incubate 30 s, and discard
•  Pipette again to remove all EtOH
•  Air Dry for 2-5 minutes
•  Remove from magnet and Add 42 ul low EDTA TE to each sample
•  Pipette mix 10x or until beads are resuspended
•  Incubate 5 minutes at RT
•  Return tubes to magnet for 2 minutes
•  Remove and save 40 ul of supernatant in new tubes and place on ice

cDNA QC of remaining 2 ul

•  Add 9 ul ultra pure water to remaining 2 ul and mix without disturbing beads
•  Incubate 1 minute
•  Transfer 10 ul to new PCR tubes and place on ice
•  Prepare QC MM:

•  Add 15 ul of QC MM to each 10 ul sample
•  Run on Thermocycler:

HOLD POINT 4 C in Thermocycler overnight

QC bead cleanup

•  Stage Reagents at RT
•  CE Buffer
•  Illumina Purification Beads >20 minutes
•  ultra pure water
•  low EDTA TE
•  tapestation supplies
•  Prepare at least 400 ul of fresh 85% Ethanol per rxn (340 ul EtOH and 60 ul ultra pure water)
•  Add 15 ul ultra pure water to each sample
•  Add 32 ul Illumina Purification Beads, Mix by pipette 15 times with 67 ul
•  Incubate 5 minutes RT
•  Place on Magnet, Incubate 5 minutes
•  Discard ~72 ul supernatant
•   Add 200 ul 85% EtOH, incubate 30 s, and discard
•  Add 200 ul 85% EtOH, incubate 30 s, and discard
•  Pipette again to remove all EtOH
•  Air Dry for 2-5 minutes
•  Remove from magnet, Add 11 ul low EDTA TE, Mix by pipette >10X
•  Incubate 5 minutes RT
•  Return tubes to magnet for 2 minutes
•  Remove and save 10 ul supernatant for QC
•  Run samples on HS D5000 ScreenTape

Library Prep

•  Stage Reagents at RT
•  Illumina Purification Beads >20 minutes
•  ultra pure water
•  low EDTA TE
•  Thaw Reagents on ice
•  Library Prep Buffer
•  Library Prep Enzymes
•  Library Prep Mix A
•  Library Adapter Mix
•  4 X PCR MM
•  UDI Library Index Mix Strip
•  Obtain the 40 ul of cDNA and place on ice
•  Pre-Chill Thermocycler
•  Add 10 ul MM to each tube:

•  Vortex to mix 5-10s
•  Thermocycle with lid at 105C:

•  Prepare Library Adapter Mix

•  Add 5 ul to each rxn
•  Pipette mix Library Prep Mix A 15X at ~130 ul and place on ice
•  Add 20 ul to each sample (viscous)
•  Pipette Mix 10X at 40 ul, brief centrifuge to collect
•  Incubate 20 C for 20 minutes (no heated lid)
•  Prepare at least 400 ul 85% EtOH per sample
•  Resuspend Illumina Purification Beads by vortex < 30s
•  Remove ligation rxn from incubation
•  Add 60 ul Illumina Purification Beads to each sample, pipette mix
•  Incubate 5 minutes at RT
•  Place on magnet 5 minutes
•  Remove ~135 ul supernatant
•  Add 200 ul 85% EtOH, incubate 30 s, and discard
•  Add 200 ul 85% EtOH, incubate 30 s, and discard
•  Pipette again to remove all EtOH
•  Air Dry for 2-5 minutes
•  Remove from magnet and add 34 ul ultra pure water, Pipette mix >10X at 32.5 ul
•  Incubate 5 minutes at RT
•  Return tubes to magnet for 2 minutes
•  Transfer 32.5 ul to new 0.2 ml tubes, store on ice
•  Thaw 4X PCR MM and UDI Index Mix strip
•  Add 5 ul Unique UDI Library Index Mix to each tube and record
•  Add 12.5 ul 4X PCR MM
•  Pipette Mix 10X at 32ul
•  Thermocycle with lid at 105C:

HOLD POINT 4 C in Thermocycler overnight

•  Stage Reagents
•  Illumina Purification Beads >20 minutes
•  Prepare at least 400 ul 85% EtOH per sample
•  Resuspend Beads by vortex
•  Add 45 ul to PCR rxns
•  Add 76 ul Illumina Purification Beads to each
•  Mix by pipette 15X at 160 ul
•  Incubate 5 minutes at RT
•  Place tubes on Magnet for 5 minutes
•  Discard ~172 ul supernatant
•  Add 200 ul 85% EtOH, incubate 30 s, and discard
•  Add 200 ul 85% EtOH, incubate 30 s, and discard
•  Pipette again to remove all EtOH
•  Air Dry for 2-5 minutes
•  Remove tube(s) from magnet, Add 21 ul low EDTA TE, Mix pipette 10X 20ul
•  Incubate 5 minutes at RT
•  Return tubes to magnet for 2 minutes
•  Transfer 20 ul supernatant to new tubes

HOLD POINT -20 C long term

•  Tape Station Each library
•  qPCR or Qubit each library