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  • Bruno Vellutini 11:41 on 2014/06/05 Permalink
    Tags: , , rna   

    RNA extraction of Lineus longissimus embryos and juveniles 


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    Starting extraction with snap-frozen samples of different stages of Lineus longissimus with the TriReagent protocol.

    # stage date ng/µL 260/280 260/230 total ng
    1 late cleavage 0d 02/04/14 204.0 1.89 1.83 4794.0
    2 blastula 1d 03/04/14 189.2 1.88 1.98 4446,2
    3 gastrula 2d 04/04/14 182.3 1.88 2.15 4284,05
    4 pilidium 3d 05/04/14 319.6 1.92 1.75 7510,6
    5 pilidium 5d 07/04/14 158.8 1.85 1.27 3731,8
    6 juvenile 2 months 12/05/14 50.3 1.77 1.03 1182,05
    7 juveniles 3 months 13/06/14 41

    Since the samples of the juveniles look so bad I think I’ll do another extraction.

     
  • Bruno Vellutini 16:29 on 2013/10/21 Permalink
    Tags: , , rna   

    Priapulus profiling in situ matching 


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    I got some in situ pictures from Priapulus and  tried to match them to the staged expression levels data. They are congruent more or less, although it is not so straightforward since stage pictures is not complete (early/late missing) and embryos are not exactly the specific rna stages.

    However, I can see gastrulation as a major burst of expression for most of the genes we have the in situs.

    bcatenin brachyury cdx evx fgf8 foxa1 foxq2 fz58 gata456 gsc

     
  • Bruno Vellutini 10:52 on 2013/10/10 Permalink
    Tags: , , rna   

    Priapulus profiling initial 


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    Screenshot 2013-10-10 09.38.45

    I ran a basic RSEM analysis during the weekend to calculate the expression levels for Priapulus stages. Now I’m following the stepsfor the visualization of the results. The photo above shows the expression levels of the same for different stages. I’m converting WIG files exported with RSEM to TDF to be visualized in IGV.

     
  • Bruno Vellutini 22:07 on 2013/09/01 Permalink
    Tags: , , rna   

    Priapulus RNAseq profiling, first assemblies 


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    I just finished the assemblies with Agalma. Do you mind to have a look at the FastQC reports? They mostly fail on “Per base sequence/GC”, “Overrepresented sequences” (some adaptors and primers), and “Kmer content”. I also included the Agalma reports. Reports are linked in the Assembly section for each sample:

    https://dl.dropboxusercontent.com/u/203439/Priapulus_caudatus/Methods.html

    I also included a comparison with CLC assemblies I did in the mean time (last table). They seem worst than Agalma (shorter contigs).

     
  • Bruno Vellutini 16:14 on 2013/08/07 Permalink
    Tags: , , rna   


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    Priapulus sequences have arrived! Located at “Hexabackup/Animals/Priapulus_caudatus/RNAseq_profiling/”. Unfortunately one of the stages failed Pc1_7d.

    Screen Shot 2013-08-07 at 4.11.04 PM

     
  • Bruno Vellutini 20:37 on 2013/05/04 Permalink
    Tags: , , rna   

    RNA extraction for 2nd series of Priapulus stages and L. longissimus 


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    Starting an extraction for another series of Priapulus stages. Using Phase Gel Lock tubes, LPA carrier and overnight precipitation.

    Embryos were at the bottom so it was a little easier to pick, but in the end I had to spin them many times to dry out the collecting tube to 100 µL. Another problem was using 1 mL for separating the phases in the gel lock tubes. They only support 750 µL. For this reason the gel was at the top in most tubes. So I collected the aqueous phase with Chema’s help and mixed an equal volume of chloroform to take the phenol out. Only after this we added the carrier (1 µL) and let it precipitate overnight.

    05/05/13

    Some tubes had a transparent bottom phase. After spinning this unknown droplet trapped the pellet, nothing changed after the ethanol wash. When I added 22 µL of DEPC water the pellet and droplet seem to have dissolved. In the tubes where there was a clear and nice pellet a solid case remained where the pellet was. These are the results of the NanoDrop:

    Sample ID ng/ul 260/280 260/230
    Pc2 oocytes 106,74 1,78 0,41
    Pc2 1d 186,70 1,95 0,38
    Pc2 2d 236,72 2,18 0,50
    Pc2 3d 246,45 2,04 0,50
    Pc2 4d 834,65 1,93 0,43
    Pc2 5d 215,32 2,13 0,43
    Pc2 6d 112,33 1,68 0,39
    Pc2 7d 303,38 1,85 1,37
    Pc2 9d 283,21 2,04 0,55
    Pc2 12d 390,20 1,92 0,52
    Pc2 15d 485,06 1,94 0,96
    Pc2 2nd larvae 131,70 1,74 0,40
    Llon mixed 288,17 1,85 0,59

    Candidates for re-extraction are: oocytes, 7d, 2nd larvae

     
  • Bruno Vellutini 13:11 on 2013/05/03 Permalink
    Tags: , rna   

    Re-extraction of Priapulus stages 


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    Re-extracted a couple of samples from Priapulus stages using gel-lock, carrier, and overnight precipitation and here are the curves.

    For the 4d sample the curves are the same, just lower.

    5d sample looks better with a lower 270 and 230 peaks although the 260/280 ratio is the same. 260/230 has improved.

    Concentration was lower in both.

    So, I’ll do the extraction for the second series using the gel-lock, carrier and o/n precipitation. If these curves look much better than the first series than it might be worth to re-extract them all. But maybe not..

     
  • Bruno Vellutini 20:00 on 2013/04/30 Permalink
    Tags: , rna   

    Priapulus RNA extraction of specific stages 


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    Samples are neutrally buoyant with RNAlater, which makes picking a pain. I added 1x PBS with DEPC water to make the embryos sink, but still had to spin them at least 3 times at 8rpm to get all of them. Also, they stick along the wall making it hard for pipeting. In addition I spin the RNAse free tube with samples to dry out until maximum of 100µL, less when possible. Afterwards the protocol went ok.

    However, after centrifuging the isopropanol added sample a rock solid cement was fixed in the bottom of the tube. Pellet like flakes were visible with the ethanol wash and I saw them dissolving with the addition of DEPC water.

    These were the NanoDrop measurements:

    Sample ID ng/ul 260/280 260/230
    Pc oo 152,94 1,65 0,47
    Pc 1d 106,72 1,49 0,48
    Pc 2d 306,38 1,71 0,63
    Pc 3d 264,63 1,64 0,58
    Pc 4d 218,24 1,71 0,53
    Pc 5d 225,42 1,64 0,55
    Pc 6d 167,43 1,57 0,51
    Pc 7d 81,00 1,55 0,58
    Pc 9d 249,42 1,56 0,58
     
  • Bruno Vellutini 13:51 on 2013/04/17 Permalink
    Tags: , rna   


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    Picked last dish of Lineus longissimus larvae with ~3 weeks old to RNALater.

     
  • Bruno Vellutini 16:46 on 2013/04/09 Permalink
    Tags: , rna   


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    I fixed in RNALater the second dish with Lineus longissimus larvae. I had to do an extra cleaning to get rid of super tiny bacteria/protozoa (?). So this is the sample ~1 week after spawning.

     
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