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Forum: Illumina/Solexa 05-31-2017, 01:50 AM
Replies: 1
Views: 718
Posted By seqgirl123
Urgent - MiSeq 12.5pM PhiX concentration

If I use the MiSeq v3 kits, according to the denature & dilute guide (attached), I dilute both my PhiX and library concentration to the same concentration, and spike in the desired Phix percentage....
Forum: RNA Sequencing 12-10-2016, 08:23 AM
Replies: 2
Views: 1,307
Posted By seqgirl123
scRNA-Seq

Does anyone have a workflow or knows of a kit that allows for whole transcript single cell RNA Seq with reverse transcriptase barcoding of each cDNA, and also UMI labeling? We are currently...
Forum: Bioinformatics 12-10-2016, 08:09 AM
Replies: 15
Views: 7,561
Posted By seqgirl123
Does anyone have a workflow yet for scRNA that...

Does anyone have a workflow yet for scRNA that allows RT barcoding and UMI labeling on sequencing whole transcript RNA? If so, what kit and analysis tools do you use?
Forum: Sample Prep / Library Generation 12-07-2016, 11:57 AM
Replies: 4
Views: 2,035
Posted By seqgirl123
Hello, I have found a paper here...

Hello,

I have found a paper here http://www.nature.com/nmeth/journal/v11/n2/full/nmeth.2772.html that the supplementary table 2 "list of custom oligonucleotides" they published the full list of...
Forum: Sample Prep / Library Generation 12-05-2016, 05:49 AM
Replies: 4
Views: 2,035
Posted By seqgirl123
cmbetts, thank you for your reply. I will be...

cmbetts, thank you for your reply. I will be adding ERCC to the lysis mix in single cells. I prefer ERCC too. Do you know where I can find how to dilute ERCC into single cell lysis reactions?
...
Forum: Sample Prep / Library Generation 12-03-2016, 10:32 AM
Replies: 4
Views: 2,035
Posted By seqgirl123
Smart-Seq v4 ultra low RNA kit

Hello,

I would be using this kit for single cell RNA in a 96 well plate. I will be preparing the reactions in a plate. I would like to know if anyone has spiked in ERCC or Array Control RNA...
Forum: Sample Prep / Library Generation 10-03-2016, 08:11 AM
Replies: 0
Views: 449
Posted By seqgirl123
Fluidigm C1 blocking reagent

I would like to know the components of the Fluidigm C1 blocking reagent if anyone has it? They provide so much of the other reagents and not enough of this, sometimes if I need to rerun a chip, I run...
Forum: Sample Prep / Library Generation 10-03-2016, 08:06 AM
Replies: 6
Views: 2,156
Posted By seqgirl123
Have you been able to resolve this issue? I would...

Have you been able to resolve this issue? I would try each of the following: 1) use a different RT, 2) clean up the cDNA with SPRI beads (you can size select the cDNA) and proceed into library prep,...
Forum: Sample Prep / Library Generation 04-12-2016, 09:29 AM
Replies: 3
Views: 2,212
Posted By seqgirl123
Have you commercialized this yet? Our lab has...

Have you commercialized this yet? Our lab has interest in small RNA spike in controls. Please let me know where to access these.
Forum: Sample Prep / Library Generation 10-16-2015, 10:11 AM
Replies: 2
Views: 817
Posted By seqgirl123
Do you use the C1 NextGenSeq? Fluidigm gives the...

Do you use the C1 NextGenSeq? Fluidigm gives the illustra WGA kit as their choice. When using that, it doesn't work to evenly amplify the template. Does anyone use another kit than the one Fluidigm...
Forum: Sample Prep / Library Generation 10-15-2015, 08:03 AM
Replies: 2
Views: 817
Posted By seqgirl123
Fluidigm C1 for DNA-Seq protocol

Does anyone use Fluidigm C1 instrument on cells using DNASeq script and final library prep for NGS? Our lab has followed their published protocol using a control cancer cell line & we haven't got...
Forum: Sample Prep / Library Generation 11-08-2014, 06:46 PM
Replies: 1
Views: 1,147
Posted By seqgirl123
Kapa library quantification kit

Has anyone had issues with the kapa library quantification kit? I use it often and recently noticed I'm getting high ct values even in my control libraries. Do you leave the kit to thaw to room...
Forum: Sample Prep / Library Generation 09-05-2014, 06:31 AM
Replies: 268
Views: 102,170
Posted By seqgirl123
Is anyone quantitating the amount (ng) of the...

Is anyone quantitating the amount (ng) of the total RNA in your samples prior to using these single cell kits? Is it necessary for these kits, especially the Fluidigm?
Forum: Sample Prep / Library Generation 08-28-2014, 01:18 PM
Replies: 1
Views: 1,563
Posted By seqgirl123
Agilent XT2 kit

I can't use the XT2 blocking oligos because my libraries are 6nt indices and Agilent XT2 only support 8nt indices. I'm thinking of purchasing the Xgen blocking oligos.

Has anyone tried using the...
Forum: General 08-19-2014, 05:47 PM
Replies: 16
Views: 3,836
Posted By seqgirl123
I believe the genetics is there underlying sexual...

I believe the genetics is there underlying sexual orientation. I knew when I was 4years I was drawn to the opposite sex. It was a no brainer to me. And I wasn't exposed to the social 'dating' cues...
Forum: General 08-19-2014, 07:47 AM
Replies: 0
Views: 1,336
Posted By seqgirl123
cost of Nanostring

I'm interested in knowing what is the cost of Nanostring these days? I found one article listing price at $235,000 back in 2012. Has it gone down or still the same? I can't reach anyone on the phone...
Forum: Illumina/Solexa 06-25-2014, 05:16 PM
Replies: 2
Views: 1,253
Posted By seqgirl123
insert size for sequencing

I have a genome library at 350bp. I sequenced it using 2x150bp read length on Miseq v2. Will I miss out on information because the insert size (350bp) is larger than the total sequencing read length...
Forum: Sample Prep / Library Generation 06-19-2014, 05:47 PM
Replies: 1
Views: 2,307
Posted By seqgirl123
mRNA fragmentation RNA Seq

I am doing RNA-Seq, of mRNA by polyA bead selection. In the protocol I am using, it says to heat treat the mRNA for fragmentation. It does heat treatments at 94C at varying time lengths for varying...
Forum: Illumina/Solexa 06-19-2014, 03:34 PM
Replies: 1
Views: 1,965
Posted By seqgirl123
In the future, you should be able to contact the...

In the future, you should be able to contact the vendor and request them to ship you extra. They will do this if you run out of reagent in the middle of your experiment or accidentally use the wrong...
Forum: General 10-29-2013, 07:53 AM
Replies: 2
Views: 1,941
Posted By seqgirl123
coverage question

What is the depth of coverage on human genome for a HiSeq rapid run versus high output? for a miseq, what is the depth of coverage that can be done on it? thanks in advance. on the older hiseq 2000...
Forum: Illumina/Solexa 10-24-2013, 12:29 PM
Replies: 4
Views: 2,096
Posted By seqgirl123
flowcell help

Can a single end library be sequenced on a paired end flowcell? Or is it a paired end library can be sequenced on a single end flowcell?

For the dual index libraries, Illumina says one can...
Forum: Sample Prep / Library Generation 04-15-2013, 07:24 AM
Replies: 2
Views: 4,232
Posted By seqgirl123
Ampure XP beads cleanup and concentration

Does anyone know what ratio of Ampure XP beads:sample I must use to remove material from my library that is 150bp and below? My library is in 30ul of 1x TE.

Also has anyone ever concentrated...
Forum: Sample Prep / Library Generation 04-14-2013, 05:37 PM
Replies: 1
Views: 5,497
Posted By seqgirl123
nextera kits

I am slightly confused with all the different Nextera kits being sold by Illumina. I feel like there are too many, or even within a kit, there are too many boxes.

For my purposes, I'd like to...
Forum: Sample Prep / Library Generation 03-28-2013, 06:03 PM
Replies: 1
Views: 1,963
Posted By seqgirl123
Illumina sequences with Kapa kit

Has anyone used Illumina Truseq adapters and the PPC primer cocktail with the Kapa library preparation kit (Illumina compatible version)? The Kapa kit allows the user to use outside adapter and PCR...
Forum: Sample Prep / Library Generation 04-27-2011, 06:22 PM
Replies: 3
Views: 2,096
Posted By seqgirl123
shearing question using Covaris

Would like to understand something when I use Covaris for shearing.

I have DNA that has been diluted to 5ug in 120ul for shearing or 41ng/ul. I shear and measure concentration by Picogreen. But...
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