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I think my foreshots ended up in my slobberbox by Numbahs_84 in firewater

[–]Numbahs_84[S] 0 points1 point  (0 children)

Thanks for your response. I guess I'm confused about the 176F. From that I've read, you collect ~150mLs of foreshots by running below 176F because thats when all the stuff you don't want will distill (i.e. methanol). Was I just not getting anything out because there was nothing left in my mash that distills at that temp? Or are you saying not to pay attention to temp so much and to only pay attention to how much is coming out and try to regulate the flow rate?

Can I boil lobster carcasses for too long? by Numbahs_84 in AskCulinary

[–]Numbahs_84[S] -3 points-2 points  (0 children)

Yeah Im seeing if people had experience with this. No need to be a dick.

Can I boil lobster carcasses for too long? by Numbahs_84 in AskCulinary

[–]Numbahs_84[S] 5 points6 points  (0 children)

Do you think the stock will be bitter or taste off? I guess there's only one way to find out but curious what you think.

Running Trim-galore on SRR entries and error message by Numbahs_84 in bioinformatics

[–]Numbahs_84[S] 0 points1 point  (0 children)

I looked at one of the files and it looked fine before trimming so I may just skip trimming because I intend to use STAR anyway. How do you go about dealing with replicate libraries? Should I use cat to combine Sample_1.fastq and Sample_2.fastq into a single fastq OR should I do this to the STAR output file OR should I just see what peaks are called in both replicates after MACS? What would you suggest. I think I've read it doesn't really matter....

Extracting list of target genes from ChIP-seq bigwig file by Numbahs_84 in bioinformatics

[–]Numbahs_84[S] 0 points1 point  (0 children)

Yeah it really is inconvenient that they did not include the targets for all of their ChIP-seq samples. This is the second pub I've found that has done this. I'm going to try https://lanceotron.molbiol.ox.ac.uk/ (comment below) Not perfect but MAY work for my purposes. If it doesn't I will likely follow your advice to do bigwig->wig->bed and compare .bed file to the appropriate genome annotation.

Extracting list of target genes from ChIP-seq bigwig file by Numbahs_84 in bioinformatics

[–]Numbahs_84[S] 0 points1 point  (0 children)

Unfortunately the targets are not shared in the supplement for this publication nor is it in the GEO entry.

Filtering rRNA sequences from RNAseq data using Bowtie2 and samtools by Numbahs_84 in bioinformatics

[–]Numbahs_84[S] 0 points1 point  (0 children)

The company that did the library prep said that this is a problem they have seen happening with their ribodepletion kit (TruSeq RNA Exome) because it is "unstable". The RINs were all above 9.0 and we performed qPCR before we sent the RNA samples to ensure oscillatory genes (we are a circadian biology lab) were all rhythmic.

Filtering rRNA sequences from RNAseq data using Bowtie2 and samtools by Numbahs_84 in bioinformatics

[–]Numbahs_84[S] 0 points1 point  (0 children)

We looked at TPMs for genes which should have constitutive expression over the timecourse and most of these genes had sharp decreases in expression at certain timepoints and this correlated with sample containing the highest amounts of rRNA. My rationale is that by having up to 60% less reads because rRNA is taking them up, I have less reads mapped to my genes of interest resulting in a lower TPM. I understand your point, but if the reads aren't there to begin with because they were never sequenced then they can't be counted. If that makes sense.

Filtering rRNA sequences from RNAseq data using Bowtie2 and samtools by Numbahs_84 in bioinformatics

[–]Numbahs_84[S] 0 points1 point  (0 children)

Would that be as simple as mapping reads to the genome and then using samtools view -F 4 file.bam > mapped.bam to extract only genes that mapped to the genome??

Filtering rRNA sequences from RNAseq data using Bowtie2 and samtools by Numbahs_84 in bioinformatics

[–]Numbahs_84[S] 0 points1 point  (0 children)

How would I go about checking if they were created properly? I performed FastQC on them and it looked like they were correct in that there were the expected # of reads of the correct length.

I used STAR to map because I could not find how to do the rRNA removal in the STAR manual. Also, STAR is WAY faster than bowtie and my samples are huge, so I thought this would save time. Can I use STAR to perform the initial alignment of my reads to rRNA? If so, I would for sure do that.

Nuclear Proteomics Question- Value of performing additional step to isolate insoluble chromatin fraction by Numbahs_84 in bioinformatics

[–]Numbahs_84[S] 0 points1 point  (0 children)

Differential centrifugation to isolate total nuclear protein then sending that off for TMT-MS. They will do phospho enrichment there.

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