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Old 05-05-2019, 01:30 AM   #1
MarkiMark
Junior Member
 
Location: Tel Aviv

Join Date: May 2019
Posts: 2
Smile Human Fibroblasts

Hi all,

Wanted to ask for suggestions-comments-opinions regarding this experiment;

We are planning to sequence total RNA from 30 samples of human fibroblasts (literature points out that this type of cells are quite noisy).
We are looking for DE genes, splicing variants and even novel isoforms.

For that, total RNA will be extracted using the mirNeasy kit and then divided to two sequencing assays;

1. Transcriptome sequencing, library construction by Illumina TruSeq stranded Total RNA library (RiboZero Gold rRNA removal). Run: NovaSeq 2X150bp, 60M reads per sample (=30M paired read per sample)

2. smRNA sequencing, library construction using Illumina TruSeq small RNA library construction. Run : Hiseq 2500 rapid run mode, 1X50bp, 125M reads per lane (10-12 samples per lane).

There will be no biological replicates due to passage limitations.
After a few considerations, we have decided not to sequence technical repeats.
We are aware that 60M reads per sample may not be sufficient for novel isoforms, but budget limitations exist.

Many Thanks,

Mark
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