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=Experiment: Increase # of wash cycles between hybridization and ligation to improve specificity= ==Background== *[[Matt:LabNotes/2016-12-5|DART-FISH with SplintR using probe set with unused barcodes and stained with Anti-NeuN after]] revealed **high number of decoded rolonies with unused barcodes **'''many glia-specific rolonies in cells stained with anti-NeuN''' ***this is very problematic for using rolony gene markers as a way to classify cell types ***probably due to low specificity (padlock probe hybridization and/or ligation) ****hybridization is carried out at 55C for ~20hr ****sample was then washed twice with 55C PBS ****then hybridized probes were ligated at 37C by SplintR for 20min ***my hypothesis is that falsely ligated probes are due to incomplete removal of non hybridized probes during wash step ****by increasing washes should see decrease in false positives ==Experimental Design== *Use IDT ordered padlock probes to capture RNA of housekeeping genes and neuron-specific genes (RELN) *Add all probes to 3 samples *Each sample is washed different number of times (2, 6, 15) *Rolonies are imaged and then samples are stained with DRAQ5 and Anti-NeuN *Check percentage of RELN not in Anti-NeuN stained cells ==Padlock Probes== ===Housekeeping padlock probes=== #ppMALAT1_dc1 dc1-488 /5Phos/TTTCTGCCTTTACTTATCAATTC CTTCAGCTTCCCGATATCCGACGG G TCTTGCGTGCGATACGGAGT A AATGGAGGTATGACATATAATCT #ppMALAT1 dc2-Cy3 /5Phos/TTTCTGCCTTTACTTATCAATTC CTTCAGCTTCCCGATATCCGACGG T CTACTTCGTCGCGTCAGACC A AATGGAGGTATGACATATAATCT #ppACTB dc0-Cy3 /5Phos/CTGTGCTCGCGGGGCG CTTCAGCTTCCCGATATCCGACGG A CGTATCGGTAGTCGCAACGC AGGCAAAGGCGAGGCT #ppRAB7A dc1-488 /5Phos/GAAGCGAGAAGGTCCAAGTTCTG CTTCAGCTTCCCGATATCCGACGG G TCTTGCGTGCGATACGGAGT AGAGGAGACTAAACGGAGGACA ===RELN padlock probes=== *Name dcProbe Probe T4count SplintRcount Ampligasecount *ppRELN_1 dc5-Cy3+dc6-488 RELN_AAAAGAGGTTGTTTCCACTAGAAAGGATTCCACACTTTCGAGATGGTTTCC 43 67 123 */5Phos/ ATTCCACACTTTCGAGATGGTTTCC CTTCAGCTTCCCGATATCCGACGG ACGTCTGCGTACCGGCTTAG CACGCTTACGATCCCGCTAT AAAAGAGGTTGTTTCCACTAGAAAGG *ppRELN_2 dc5-Cy3+dc6-Cy3 RELN_CTCTGTGATGCCTGAACACTTGTAGATGTGTATAGTCCTGTCACCAGCAA 19 31 70 */5Phos/ ATGTGTATAGTCCTGTCACCAGCAA CTTCAGCTTCCCGATATCCGACGG ACGTCTGCGTACCGGCTTAG TCGTAACCCGTGCGAAGTGC CTCTGTGATGCCTGAACACTTGTAG *ppRELN_3 dc5-Cy3+dc6-Cy5 RELN_GAAAATTCCAGTCTCACTGGATCCGCGGATGAGCTATCAGTCGAACAGC 70 11 20 */5Phos/ CGGATGAGCTATCAGTCGAACAGC CTTCAGCTTCCCGATATCCGACGG ACGTCTGCGTACCGGCTTAG CTCTCGTAGCGTGCGATGAG GAAAATTCCAGTCTCACTGGATCCG ==Protocol== *3 Samples: GTEx Patient 5342 Occipital Cortex sectioned by Yun on 11-22-2016 *5% Gel mix and protocol is same as [[Matt:LabNotes/2016-6-28#Gel_Mix|this with Acryloyl-X]] {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | width="51" height="14" | Component | width="51" | Volume |- style="font-size:11pt" valign="bottom" | height="14" | 40% AB 199:1 Mix | align="right" | 6.25 |- style="font-size:11pt" valign="bottom" | height="14" | 10% BSA | align="right" | 1 |- style="font-size:11pt" valign="bottom" | height="14" | 10mg/ml Acryloyl-X, SE in DMSO | align="right" | 1 |- style="font-size:11pt" valign="bottom" | height="14" | H2O | align="right" | 39.75 |- style="font-size:11pt" valign="bottom" | height="14" | 5% TEMED | align="right" | 1 |- style="font-size:11pt" valign="bottom" | height="14" | 5% APS | align="right" | 1 |- style="font-size:11pt" valign="bottom" | height="14" | Total | align="right" | 50 |} ===Day 1=== #Prepare 3 plastic culture dishes with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate #*EtOH and UV sterilize #Made 4% PFA in 1X PBS #*10ml 16% PFA + 4ml 10X PBS + 26ml H2O #Take out brain section from -80C and dry on 50C hot plate for 3min #Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C #Wash twice with cold 1X SSPE by submerging #Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT #Wash with cold nf-H2O three times '''and check for degradation''' #Skip pepsin step usually done here #Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue #*Secure in plastic jig #Add 50ul gel casting mix '''filtered and degassed''' #Seal in plastic bag and vacuum out air before filling with argon #Let sit at RT for 30min #Attach coverslip to bottom of petri dish #Aspirate non-polymerized gel and wash twice with 1X PBS #Prepare SplintR Mix #*1ul of each 100uM probes (4) and 0.5ul of each 200uM probes (2) + 595ul H2O for 1uM total conc #*In SplintR Mix each probe has concentration = 100nM {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X SplintR Buffer||10 |- | Padlock Probe Mix 1uM||60 |- | H2O||30 |- | Total||100 |} #Move sample to 60C HybEZ oven and slowly decreases to 55C and held for ~2hr #*Only 2 samples because 1 cracked #*The 2 samples were left at 4C in 1X PBS for 1 or 2 hrs before adding SplintR Mix ===Day 2=== #Wash with 1X PBS preheated to 55C with sample on 55C hot plate #*Wash 2x for sample #1 #*Wash 10x for sample #2 #Add SplintR Enzyme and incubate at 37C for 20min #*20ul 10X Buffer + 6ul SplintR + 174ul H2O #Wash with 1X PBS twice #Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) pre-heated to 65C and incubate 1hr at 55C #Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS #Prepare RCA reaction mix on ice {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||174 |- | 10X Phi29 Buffer||20 |- | 25mM dNTP||2 |- | 4mM aa-dUTP||2 |- | Phi 29 (low conc)||2 |- | Total||200 |} #Add RCA mix and incubate at 30C overnight (~15hrs) ===Day 3=== #Wash with 1X PBS twice (normally once) #Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr #Wash with 1X PBS twice #Add 1M Tris pH 8.0 and incubate at RT for 30min #Wash with 1X PBS twice ==Imaging== ===Settings=== *20X objective *2048x2048 px *4x line average *3 positions in each dish ===Rolony FISH=== #Add 0.5uM dc5_Cy3 in 30% formamide + 2X SSC preheated to 75C (detects RELN) #Incubate for 10min at RT #Wash with 2X SSC twice #Find positions near layer 1 of cortex with RELN and image #Strip twice with 15min incubation of 80% formamide preheated to 75C #*Wash with 1X PBS twice after each strip #Image to confirm no signal #Add 0.5uM FISGA_Adpt_Cy3 in 30% formamide + 2X SSC preheated to 75C (detects all) #Wash with 2X SSC twice #Image same positions ===Anti-NeuN Staining Procedure=== #Rinse tissue with 1X PBS for 30min #Incubate section for 30min in 1ml blocking buffer #*50ul DKS + 250ul 10% BSA + 0.1ul TX-100 + 100ul 10X PBS + 600ul H2O #Incubate section for 1hr in 1ml NeuN antibody solution #*1ul rabbit anti-NeuN (Millipore MABN140) diluted in 999ul blocking buffer same as in step 2 #Wash 3x 5min in 1X PBS #Stain with Donkey anti-Rabbit Alexa 488 for 1.5hr #*DxR488 stock was diluted 1:200 in blocking buffer without the TX-100 #Wash 3x 5min in 1X PBS #*2nd wash use 5uM DRAQ5 in 1X PBS #*3rd wash use 1X PBS + 0.1% Tween-20 #Add 150ul Vectashield mounting media with antifade and allow 10min before imaging ===DRAQ5 Staining=== #Incubate in 5uM DRAQ5 + 1X PBS for 15min and don't wash before imaging again
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