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Forum: RNA Sequencing 07-30-2018, 07:10 AM
Replies: 12
Views: 3,227
Posted By alekzs
Yes... I used RSEM for the counting and the index...

Yes... I used RSEM for the counting and the index generation with the ERCC-appended hg19 file had failed because the chr-labels weren't compatible so RSEM used an old index without ERCC genes.
I...
Forum: RNA Sequencing 04-27-2018, 12:43 PM
Replies: 12
Views: 3,227
Posted By alekzs
ha, that approach was far easier... grep...

ha, that approach was far easier...

grep "ERCC-" 10B02_3.sam -c
6770

So, yes... they are there, just don't end up in any count file.
Forum: RNA Sequencing 04-27-2018, 12:30 PM
Replies: 12
Views: 3,227
Posted By alekzs
Tail of FASTA file: >ERCC-00171 DQ854994...

Tail of FASTA file:
>ERCC-00171 DQ854994 Ac03459967_a1 Ac03460063_a1...
Forum: RNA Sequencing 04-27-2018, 10:25 AM
Replies: 12
Views: 3,227
Posted By alekzs
I did everything myself so chances are 50-50 I...

I did everything myself so chances are 50-50 I guess. :D
Anyhow, even if I didn't add the spike ins, shouldn't the gene names from the reference appear in a gene count file? Like, normal genes get...
Forum: RNA Sequencing 04-27-2018, 10:07 AM
Replies: 12
Views: 3,227
Posted By alekzs
Yes, I added both FASTA and GTF annotations und...

Yes, I added both FASTA and GTF annotations und used the hybrid!
Forum: RNA Sequencing 04-27-2018, 08:33 AM
Replies: 12
Views: 3,227
Posted By alekzs
ERCC - no gene counts

Hi!
I have a problem with recovering added ERCC to my RNAseq samples. Briefly, I'm doing smart-seq2 on single human T cells and align with STAR to hg19+ERCC sequences. Both FASTA and GTF file have...
Forum: Sample Prep / Library Generation 01-05-2018, 02:07 PM
Replies: 5
Views: 1,709
Posted By alekzs
I have tried increasing the PCR cycles up to 23...

I have tried increasing the PCR cycles up to 23 but still got this double-bump pattern.
The samples were frozen, I sort directly into lysis buffer (max 5min per plate), directly spin the plate down...
Forum: Sample Prep / Library Generation 01-05-2018, 09:11 AM
Replies: 5
Views: 1,709
Posted By alekzs
Single cell RNA-seq trouble

Hi folks,
I'm trying to set up a single-cell RNAseq platform for human primary cells, so far semi-successfull. Hopefully you can give me some feedback on how to optimize and fix the problems.
...
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