Tagged: Priapulus caudatus Toggle Comment Threads | Keyboard Shortcuts

  • Bruno Vellutini 16:18 on 2014/01/21 Permalink
    Tags: , phylogenomics, Priapulus caudatus, transcriptome   

    Dunn Lab – Brown University – Winter 2014 

    From 20/01/2014 to 14/02/2014 I’m staying at Casey Dunn’s Lab to analyze RNAseq data from priapulid development and acoel phylogenomics.

    Everything I’m doing is documented on GitHub: https://github.com/nelas/bcv-at-brown

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

    Priapulus profiling in situ matching 

    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: , Priapulus caudatus,   

    Priapulus profiling initial 

    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: , Priapulus caudatus,   

    Priapulus RNAseq profiling, first assemblies 

    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: , Priapulus caudatus,   

    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: , Priapulus caudatus,   

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

    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: Priapulus caudatus,   

    Re-extraction of Priapulus stages 

    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: Priapulus caudatus,   

    Priapulus RNA extraction of specific stages 

    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 22:46 on 2012/11/22 Permalink
    Tags: Priapulus caudatus,   

    Priapulus fertilization and RNA-seq time series 

    Fertilization of Priapulus brought from Kristineberg was successful. Sperm was added at 23h. I picked unfertilized +2000 oocytes into RNAlater and will continue to do this in the next 10d.

    Samples fixed: 0-7, 9d, 12d, 15d, 18d.

     
  • Bruno Vellutini 19:27 on 2012/11/13 Permalink
    Tags: , , , , , , Priapulus caudatus, , , , ,   

    Probe synthesis for Lineus et al. 

    Started a probe pcr for synthesizing the following probes of Lineus ruber, Priapulus caudatus, and Terebratalia transversa:

    Species Gene ID Probe Kit
    Lineus ruber foxa BV196 SP6
    Lineus ruber six3/6 BV199 SP6
    Lineus ruber cdx BV204 SP6
    Lineus ruber gsc BV205 T7
    Priapulus caudatus tdr BV34 T7
    Priapulus caudatus mael BV35 SP6
    Priapulus caudatus gmcl BV183 T7
    Terebratalia transversa runx2 BV166 SP6

    13/11/12

    Run gel.

    14/11/12

    Dna extraction.

    15/11/12

    Transcription reaction + precipitation.

    16/11/12

    Specs:

    Lr foxa 688,23
    Lr six3/6 383,21
    Lr cdx 303,11
    Lr gsc 501,31
    Pc tdr 3853,39
    Pc mael 97,89
    Pc gmcl 2287,30
    Tt runx2 345,39

     
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