Total RNA-sequencing

In conjunction with its Analysis Working Group and Steering Committee, GeneLab has developed the following standards for total RNA-sequencing.

Sequencing depth

GeneLab recommends the following target sequencing depths:

 

Representative genome

Transcriptome complexity

Ribodepleted RNA-seq

Human

H. sapiens GRCh38.p12

High

60 M clusters, 150 bp PE

Rodents

M. musculus GRCm38.p6

High

60 M clusters, 150 bp PE

Fruit fly

D. melanogaster Release 6 plus ISO1 MT

Medium

40 M clusters, 150 bp PE

Worms

C. elegans WBcel235

Medium

40 M clusters, 150 bp PE

Plants

A. thaliana TAIR10.1

High

60 M clusters, 150 bp PE

Fungi

S. cerevisiae S288C R64

Low

20 M clusters, 150 bp PE

Bacteria

E. coli str. K-12 substr. MG1655

Low

20 M clusters, 150 bp PE

 

Spike-in controls

At a minimum, GeneLab recommends that all sequencing samples include ERCC Spike-in Mix 1 in all samples. This will allow assessment of the dynamic range within each sample.

In addition, in experiments focused upon comparing two levels of a given factor (spaceflight vs. ground control), GeneLab recommends including Mix 1 in the level A samples (spaceflight) and Mix 2 in the level B samples (ground control). This allows direct assessment of the available power in the data.

In all cases, these Mixes should be added to samples before library preparation and sequencing.    

Mix 1 available here.
Mix 1 and 2 available here.

Library preparation and sequencing standard

GeneLab recommends that all sample pools to be sequenced include at least one replicate of the SEQC Universal Reference RNA available here (Human and/or mouse). This RNA should be included as a sample during each library preparation batch.

 

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