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==RNA-Seq of BA8 Tissue Section== *Do RNA-Seq on same tissue sections we do DARTFISH on to compare RNA-Seq to DARTFISH **This time sequence from WHOLE tissue section (approx 4cm^2) to validate protocol ***Also use this test to decide whether polydT or Random Primer Mix is better for RT **In future cut out small tissue section (approx 1mm^2) to match same region we do DARTFISH on (and maybe another small tissue section on opposite end) *Hypothesis: DARTFISH counts will correlate better with the small cut out tissue section than the whole section **This will support that DARTFISH can show regional differences in gene expression ===Isolate RNA from BA8 tissue section=== *Using ZR RNA MicroPrep kit from Zymo #Wipe all surfaces, forceps, etc with EtOH and RNase Zap #Scrape tissue off glass with a scalpel and put in 1.5ml tube #Add 400ul RNA Lysis Buffer #*Vortex 10sec and pipette with 1000p ~10x #Centrifuge at 18,000rcf for 1min #Transfer the 400ul to IIIC column and centrifuge at 8,000rcf for 30sec #Add 320ul 100% EtOH (UV'd) to flow-trhough and mix #Transfer 720ul to IC column and centrifuge at 18,000rcf for 1min #Add 400ul RNA Wash Buffer and centrifuge at 18,000rcf for 30sec #Add 30ul DNase I cocktail and incubate in 37C incubator for 15min #*1.5ul DNase I (2U/ul) + 3ul 10X Reaction Buffer + 25.5ul RNA Wash Buffer #*Centrifuge at 18,000rcf for 30sec #Add 400ul RNA Prep Buffer and centrifuge at 18,000rcf for 1min #Add 800ul RNA Wash Buffer and centrifuge at 18,000rcf for 30sec and repeat with 400ul Wash Buffer #Spin in emptied collection tube at 18,000rcf for 2min #Add 16ul H2O, let stand for 1min, and then centrifuge at 10,000rcf for 30sec *Nanodrop Measurement: 6.9ng/ul **A260/280 = 1.77 ===cDNA Synthesis=== *Using NEB ProtoScript M-MuLV First Strand cDNA Synthesis Kit and NEBNext mRNA Second Strand Synthesis Module *Divide 15ul into 3 samples **1 with d(T)VN primer (poly dT) **1 with Random Primer Mix (hexamer and d(T)VN primers) **1 Negative Control with Random Primer Mix (No RTase added to check for DNA contamination) *Followed protocol in manual including denaturing RNA at 70C for 5min and incubation at 25C for 5min for Random Primer Mix samples prior to cDNA synthesis *Stored first strand cDNA product in -20C overnight *Followed second strand synthesis protocol *Purified ds cDNA with Qiagen PCR Purification Column and eluted 30ul *Quantify with Qubit dsDNA HS kit **PolydT: Too low, out of range **RanPri: 0.225 ng/ul (x30ul) = 6.75ng **NegCtrl: Too low, out of range ====Nextera Tagmentation==== *Minimum input is ~1ng # Add 4 ul 5X Tn5 buffer to each PCR tube # Transfer 14ul of each sample to tubes # Add 2 ul Tn5 (1:50 dilution in 1:1 TE:glycerol) to each sample. Incubated 5 minutes at 55C #*Old Tn5 from Epicentre # Add 1ul Qiagen protease (20mg/ml) to each sample and incubate 15 minutes at 50C, followed by 20 minutes at 70C ====Adapter PCR==== #Make PCR master mix (per reaction): ##10 ul 3.3x NPM ##0.66 ul S511 Adapter ##0.66 ul SYBR Green #Add 21ul of Nextera sample #Add 0.66 of N7XX barcode adapters to each sample ##PolydT:N701 ##RanPri:N702 ##NegCtrl:N703 #Add 11.3 ul of PCR master mix to each sample #Incubate in thermocycler: ##72C for 3min ##95C for 30sec ##12 cycles of: ###95C for 10sec ###55C for 30sec ###72C for 30sec ##72C for 5min [[File:20150520_RNAseqBA8_Nextera.JPG|550px]] *RNA/cDNA mass was too low *Try SMART-seq next time <!-- [[File: | 650px]] # Run 5 ul sample on PAGE gel with 3 ul 6X loading dye to identify positives. ## 0.5 ul low mass ladder (Invitrogen). Equalize volume with TBE. ## 25 min @ 250 V. ## Stain 5-10 min in ~100 ml 1X TBE with 6 ul SYBR Safe. [[File:2014-06-20_RTPrimerinvitroTest_Nextera_trial2.jpg | 650px]] # Add 100 ul magnetic beads to each positive sample. Mix by pipetting and incubate 8 minutes off magnet # Place on magnet 5 minutes, then remove and discard supernatant # On magnet, wash 2x with 180 ul 80% EtOH. Let each wash sit 30 seconds removing # Let dry 5 minutes # Resuspend beads off magnet with 20 ul H2O and incubate 2 minutes, then return to magnet for 1 minute. # Extract supernatant and transfer to 0.2 ml tube *Continued on: [[Matt:LabNotes/2014-6-23]] -->
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