Murine B-cells Illumina Prep 1

• 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

Capture and Lysis

• 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