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  • Bruno Vellutini 17:17 on 2013/02/28 Permalink
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    First attempt to assemble Terebratalia transcriptome using Trinity. For now it has to be run in /sysdev directory of fritzen. First step is to remove errors in the reads. We do this by using a standalone script from ALLPATHS-LG called ErrorCorrectReads.pl. Afterwards we cut the sequencing adaptors (need to ask companies for it). Finally we run Trinity. Here are the commands for setting up and cleaning errors:

    # Setting up.
    cd /sysdev
    mkdir terebratalia && cd terebratalia
    mkdir clean cut trinity
    
    # Running.
    perl /usr/local/src/allpathslg-43869/src/paths/ErrorCorrectReads.pl PAIRED_READS_A_IN=81BF3ABXX_Terebratalia_11s002014-1-1_Hejnol_s_3_1_sequence.txt.gz \
    PAIRED_READS_B_IN=81BF3ABXX_Terebratalia_11s002014-1-1_Hejnol_s_3_2_sequence.txt.gz \
    PAIRED_SEP=100 THREADS=8 PHRED_ENCODING=64 READS_OUT=Ttra_rna.ecr > ecr.out 2> ecr.err &
    
     
  • Bruno Vellutini 01:46 on 2013/02/27 Permalink
    Tags: , results, ,   

    Engrailed slide for Japan 

    Created a segmentation slide for Andi’s talk in Japan. Tried to summarize the most relevant information about segment boundary formation in Terebratalia showing the expression of the most important genes: engrailed, wnt1, and wnt5. This is the 3 slide PDF and the first two:

    slide1_segbound_japan

    slide2_segbound_japan

     
  • Bruno Vellutini 17:20 on 2013/02/22 Permalink
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    Terebratalia fluorescent and double in situs for segmentation 

    Starting a new fluorescent and double in situ for segmentation genes. Standard procedures.

    wnt1 wnt5 dlx en (DNP)
    en + wnt1 en + wnt5 en + dlx

    23/02/13

    Probes put at 14h.

    25/02/13

    Washing day. I put the DIG-POD for all wells except en with DNP-POD (HRP).

    26/02/13

    Regular washes.

    27/02/13

    Developed first probe in Cy3 for 1h45. Stopped the reaction 2x 5 min in detergent solution at 62 °C. After washes I checked samples in the stereoscope, but could not see any clear signal in any of the samples. Doubles were blocked and incubated with second antibody (DNP-POD).

    28/02/13

    Developed second probes from doubles in Cy5. Samples were stopped in the same manner and further washed in PTw. I stained with DAPI 1:1000 in PBS for 20 min, washed 3x in PBT and stored in PTw. I mounted 1 slide with en+wnt5 and had a look under the scope.

    It seems that en with DNP-HRP has no background, but the signal was really faint. This might be do re-using the 500 µL of a 0.5 ng/µL probe (find out later that it was half diluted…).

    Snap-5163

    The wnt5 with DIG-POD resulted in a dirty and shiny larva with background all around. Cannot see any real signal. So it seems that DIG-POD needs to be washed longer or there is something wrong with the antibody or TSA kit.

    Snap-5164

    DAPI staining was ok…

    Snap-5165

     
  • Bruno Vellutini 18:32 on 2013/02/15 Permalink
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    Regular and fluorescent in situ for Terebratalia segmentation genes 

    Beginning a regular in situ for the newest wnt7 and wnt11 and for en and dlx to get their patterns. Also testing the DNP-POD for en and DIG-POD for dlx, wnt1, and wnt5. One well will be for a first double in situ with en and wnt5.

    DNP-AP >> en dlx wnt7 wnt11
    DIG-POD >> en (DNP-POD) dlx wnt1 wnt5
    DOUBLE en (DNP-POD) + wnt5 (DIG-POD)

    16/02/13

    All probes diluted to a regular concentration of 1 ng/µL and added at 14h30.

    18/02/13

    Maybe the oven was not rocking? Not sure…

    Did all the washes and added the corresponding antibodies at the following concentrations:

    Anti-Dig-AP 1:5000
    Anti-Dig-POD 1:200
    Anti-DNP-POD 1:500

    19/02/13

    Just washes.

    20/02/13

    Dig-AP started to develop at 12h00. engrailed and distal-less were already well defined in 30 minutes while wnt7 had nothing and wnt11 had a faint band in pedicle lobe. The patterns became strong after a few hours. AP substrate exchanged once and left overnight at 4°C.

    For fluorescent samples… chaos begin. I mistakenly made the TNT buffer with 1% Tween, instead of 0.1%. After developing I split the samples for en, dlx, wnt1, wnt5 into Ptw and detergent treatments. Reactions were stopped with 2x of detergent solution for 2 minutes at 60°C. I also washed the samples with the 1st POD inactivation inadvertently. At the end I see none of the expected expression patterns under the fluorescent lamp. DNP samples seem to have their mesoderm stained while Dig-POD are fluorescent in the gland cells of the epidermis.

    Will incubate the second probe overnight, anyway.

    21/02/13

    Realized I used a regular Cy3 antibody (with Anti-DNP-HRP rabbit) and not the TSA Kit Cy3… for sure this was the issue. I developed the second probe with Cy5, but now I cannot check if it worked, because the red filter is not installed in the scope.

    Ttra_mesoderm

    After developing the second probe for the doubles, the in situ worked! So engrailed-DNP does work well even for the second probe. Pictures taken under the fluorescent scope:

    en_early_larva en_larva

     
  • Bruno Vellutini 18:03 on 2013/02/13 Permalink
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    New probe synthesis for Terebratalia en and wnt7 

    Since en DNP probe had low concentration and wnt7 sequencing had failed I’m synthesizing probes for both.

    en DNP (BV208) and wnt7 DIG (BV221, actually BV215). Gel:

    2013-02-15 12.32.09

    15/02/13

    Transcription reaction.

    16/02/13

    Finishing, en DNP was again not concentrated…

    probe ng/µL
    en DNP 46
    wnt7 dig 331

     
  • Bruno Vellutini 12:44 on 2013/02/13 Permalink
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    Probe synthesis for old and new Terebratalia segmentation genes 

    Transcription reaction set for at 10h30 and precipitation at 17h00:

    ID GENE ENZYME UTP ng/µL
    BV208 en SP6 DIG 135
    BV208 en SP6 DNP 55
    BV209 dlx SP6 DIG 357
    BV211 wnt4 SP6 DIG 351
    BV218 wnt11 SP6 DIG 1234
    BV210 cdx T7 DIG 1450
    BV212 wnt8 T7 DIG 129
    BV213 eve T7 DIG 2269

    14/02/13

    Pellet washing and specs taken, see above. en was the least concentrated one, specially with DNP utp. Will need to re-do it to get more probe.

     
  • Bruno Vellutini 14:06 on 2013/02/12 Permalink
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    Re-sequencing Terebratalia Wnt7 

    Sequences of Wnt7 were crap. Re-doing them including SP6 primer.

    ID GENE COLONY NEW ID PRIMER
    BV214 Wnt7 1 BV220 T7
    BV215 Wnt7 2 BV221 T7
    BV216 Wnt7 4 BV222 T7
    BV214 Wnt7 1 BV223 SP6
    BV215 Wnt7 2 BV224 SP6
    BV216 Wnt7 4 BV225 SP6

    13/02/13

    Nice sequences now! Beginning probe PCR.

     
  • Bruno Vellutini 17:43 on 2013/02/11 Permalink
    Tags: , race,   

    RACE PCR for Wnt2 

    10/02/13

    1st round o/n.

    11/02/13

    2nd round, nothing…

    2013-02-11 15.07.09

     
  • Bruno Vellutini 17:21 on 2013/02/07 Permalink
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    PCR for remaining Terebratalia wnt genes 

    There are still 5 wnts to be cloned in Terebratalia: WntA (925bp), Wnt1b (885bp), Wnt7, Wnt11, and Wnt2 (maybe a and b). I setup a regular PCR for the first 4 using the available primers. The rest (Wnt7 and Wnt2) will be RACEd.

    08/02/13

    2013-02-08 14.44.39

    09/02/13

    2013-02-09 16.18.19

    Minipreps:

    T7
    Wnt7 1 BV173 >> BV214
    Wnt7 2 BV174 >> BV215
    Wnt7 4 BV175 >> BV216
    Wnt11 1 BV176 >> BV217
    Wnt11 2 BV177 >> BV218
    Wnt11 4 BV178 >> BV219

    10/02/13

    Sequencing PCR for all samples.

     
  • Bruno Vellutini 17:17 on 2013/02/07 Permalink
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    Transformation of plasmids for Terebratalia new probes 

    Some of the genes cloned by Yale and Andi will be used for double in situs. We have no probes and there is a limited amount of plasmid solution. So I’m transforming the plasmids again to make more minipreps (and then probes). The genes are:

    Gene ID Kit
    en TTR54 SP6
    dlx Tt-dlx-L2 SP6
    cdx TTR16 T7
    wnt4 TTR164 SP6
    wnt8 TTR104 T7
    eve Tt-eve-L2 T7

    I used 1 µL of plasmids for the transformation and diluted the bacteria solution prior to plating by 1:100 (1µL bacteria: 99µL LB at 37°C). I then plated 50 µL of the 1:100 solution at 16h30.

    08/02/13

    Perfect number of colonies!

    2013-02-08 17.42.06

    09/02/13

    T7
    en BV167
    dlx BV168
    cdx BV169
    wnt4 BV170
    wnt8 BV171
    eve BV172

    10/02/13

    Extracted the plasmids and started the probe PCR.

    11/02/13

    Probe PCR extracted.

    2013-02-11 15.07.35

     
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