Murine B-cells Illumina Prep 1

Cell 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

• 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