BioMicroCenter:RNA HTL

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For users with large numbers of eukaryotic RNA samples, The BioMicro Center offers a high-throughput RNAseq methodology that minimizes cost. High-Throughput 3' Digital Gene Expression (HT3DGE) uses a combination of molecular tagged indexes, SMARTseq chemistry and Nextera Tagmentation to produce libraries derived from the 3' ends of transcripts. SMARTseq v2 creates full length transcripts that are then tagmented and indexed using Nextera XT

LOW-INPUT STANDARD RNASEQ

Service SMARTseq v2 - high-throughput
INPUT TYPE RNA in Biorad 384w plates
use 1 quadrant.
SAMPLE BUFFER 10mM Tris pH8 (EB)
No organics
INPUT VOLUME 1uL
INPUT MASS as available. <1ng
INCLUDED cDNA generation
Library preparation
Spot check of final libraries
SUBMISSION MIT - ilabs
External - form
SUBMISSION MUST BE COORDINATED WITH BMC STAFF TO PREVENT SAMPLE LOSS. Please email biomicro@mit.edu.
UNIT 24 samples
96 samples
PLATE LAYOUT Samples should be arrayed with full columns left to right.
ADDITIONAL SERVICES AVAILABLE Sample cleaning
Sample arraying

3' DIGITAL GENE EXPRESSION

Service SMARTseq v2 - high-throughput
INPUT TYPE totalRNA in Axygen 96w plate
PCR-96-FS-C
SAMPLE BUFFER 10mM Tris pH8 (EB)
No organics
INPUT VOLUME 5uL* exact
INPUT MASS 10ng-25ng
Mass should be the same for all samples
INCLUDED cDNA generation
Library preparation
Spot check of final libraries
SUBMISSION MIT - ilabs
External - form
SUBMISSION MUST BE COORDINATED WITH BMC STAFF TO PREVENT SAMPLE LOSS. Please email biomicro@mit.edu.
UNIT 24 samples
96 samples
PLATE LAYOUT Samples should be arrayed with full rows top to bottom.
ADDITIONAL SERVICES AVAILABLE Sample cleaning
Sample arraying

The protocol is based on Soumillon et al.,doi: http://dx.doi.org/10.1101/003236 as part of a collaboration with the KI High-Throughput Screening Core. In limiting the sequence space used by the samples, fewer reads should be required for a good transcriptome.

HT3DGE uses a very early indexing step to tag each sample with a "well ID" and a molecular ID. Once tagged the samples are immediately pooled to minimize costs but failed samples cannot be easily reprepped and are not identifiable until sequencing. As such, the method is best suited for experiments where the NUMBER of samples is not limiting (material can be). The 3' nature of the protocol also limits the utility of this method in splicing applications but it should be more robust to using imperfect RNA (RIN 7+ instead of 9+ for standard RNAseq)

Analysis of HT3DGE is not a standard method supported by most open source platforms. We have built pipelines to work with this data, but working closely with the Bioinformatics team is highly recommended.

HT3DGE method.png


HT3DGE gene example.png