Wiki¶

Library prep summary¶

Sample concentration and quality was assessed by Eukaryote Total RNA Pico on Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Samples were treated with DNAse prior to library preparation. Library preparation was performed with SMARTer Stranded Total RNA (Clontech Inc, Mountain View, CA) following manufacturer’s instructions. Average final library size is between 300-400 bp. Illumina 8-nt dual-indices were used for multiplexing. Samples were pooled and sequenced on Illumina HiSeq X sequencer for 150 bp read length in paired-end mode, with an output of 80 million reads per sample.

Library prep QC¶

Sample QC reports attached. Mostly VERY low RIN scores.

Data location¶

Data is available in the form of compressed fastq files. Approximately 600 GB after unzipping the files. Files are to be uploaded onto the UCT G-drive.

Bioinformatic analyses requested¶

Standard RNA sequencing analysis including quality assessment, data normalization, alignment, gene mapping, pairwise comparisons, functional enrichment and visualization.

Papers envisaged¶

Data from this analysis will be incorporated in a manuscript phenotyping the changes in immune cells (T regulatory and Th17 cells) during infancy or as a stand-alone manuscript. The authors will include the team in the Clive Gray and Heather Jaspan group involved in this work together with the Bioinfomatician from CBIO who is willing to collaborate with this analysis.

RNAseq QC¶

Preliminary QC indicates substantial rRNA content, a very high proportion of reads to short to map as well as Illumina adapter contamination

#Stranded library
SMARTer Stranded RNA kit: https://github.com/kviljoen/RNAseq/blob/master/docs/usage.md#library-strandedness So for this library prep, see here https://chipster.csc.fi/manual/library-type-summary.html
we should use the flag --forwardStranded