Deplete human and mouse rRNA
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New! Optional gDNA removal step
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New! Hemoglobin depletion module
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Deplete human and mouse rRNA 〰️ New! Optional gDNA removal step 〰️ New! Hemoglobin depletion module 〰️
REALLY™ for RNA-SeQ
directional library prep FROM TOTAL RNA
There is a simpler workflow for your RNA-Seq needs, REALLY! Perform first-strand cDNA synthesis, library preparation and ribo-depletion in 4 simple steps, even from the most degraded inputs. Because you never generate the 2nd strand, there is no need for additional steps to retain directionality information.
REALLY rNONE™, includes our streptavidin and probe pull-down-based workflow for ribodepletion, robustly removes of ribosomal RNA and other abundant ribonucleoprotein transcripts after cDNA synthesis and adapter ligation. This reduces the time spent on processing labile RNA and giving you the option to perform downstream enrichment from either depleted or non-depleted libraries.
With REALLY hg-rNONE™ (Launched in Q1 2025), you can additionally remove the abundant hemoglobin transcript from whole blood total RNA and other input types with abundant non-informative hemoglobin transcripts.
And…our REALLYrun open-source software, takes away the guesswork from RNA-Seq data-analysis. Visit our software page to get download instructions.
products availablE
REALLY rNONE™ (rRNA depletion)
REALLY hg-rNONE™ (both rRNA and hemoglobin transcript depletion)
PAGE SECTIONS
(Scroll down or click below)
Molecular metrics
Mapping metrics
REALLY Product SPECS
Reduced protocol time by more than 3 hours in comparison to typical RNA-Seq protocols
No additional modules required to retrieve degraded samples
Input Range 10-250 ng
Optional DNaseI treatment module to remove traces of genomic DNA.
RNA converted to cDNA in the first-step, minimized exposure to RNases.
Robust depletion of cytoplasmic and mitochondrial human and mouse rRNA to < 1%
Depletion of over-abundant small RNAs - RN7SL1 and RN7SL2
Write to us at technicalsupport@claretbio.com for custom depletion probe sets.
REALLY kit components
First-strand cDNA synthesis module
SRSLY® Library Preparation Base Kit for adapter ligation and Index PCR
rNONE™ or hg-rNONE™ - Ribodepletion with or without Hemoglobin depletion module and rNONE purification beads
Clarefy™ DNA purification beads
Visit our kit component page for more information
FOR WHAT really?
APPLICATIONS
Differential expression analyses
Novel RNA discovery
Isoform analyses
RNA-Seq from low inputs
SAMPLE TYPES
Total RNA from cell-culture
RNA from fresh frozen and FFPE tissues
Whole blood derived RNA (using hg-rNONE)
How does really compare?
In a highly simplified workflow, with minimal steps, generate high quality libraries with REALLY rNONE. No 2nd strand synthesis means, fewer reagents, which mean a more cost-effective library prep! Ask us about trial kits.
REALLY™ MOLECULAR METRICS
Comparative molecular metrics of REALLY rNONE libraries vs a double-stranded library preparation method. The Agilent Universal Total Human RNA control was used as input.
With as low as 11 cycles of PCR for 10ng and only 9 cycles of PCR for inputs >50ng, REALLY rNONE generates sequence ready libraries within 7 hours. An optional UMI addition step may be performed after ribo-depletion. Write to us about downstream targeted-enrichment applications.
Comparative sequencing metrics of REALLY rNONE libraries vs a ds-Prep. Libraries were sequenced on a NextSeq500 to a depth >200M reads.
Libraries prepared with 10-250ng of the Universal Human Total RNA control (Agilent Biosciences), showed high mapping metrics, higher complexity and uniform gene body coverage for REALLY libraries in comparison to a double-stranded library preparation method that requires 2nd strand synthesis.
REALLY MAPPING METRICS?
Use lesser input in our library prep! REALLY rNONE generates libraries of higher complexity across a large input range.
Get better gene body coverage double-stranded Prep with REALLY rNONE. More uniform coverage of the transcripts allows accurate evaluation of RNA isoforms. Double-stranded approaches show a 5’ bias.
High concordance is observed between libraries. REALLY rNONE libraries had equivalent read-counts across a wide input range
HOW DOES THE HG-RNONE DEPLETION PERFORM?
REALLY libraries were prepared without depletion or with depletion (rNONE or hg-rNONE) from 10 ng and 100 ng RNA from whole blood. Libraries were sequenced to ~0.5M reads. Progressive increased mapping of unique RNA read is seen when only ribo-depletion or ribo and hemoglobin depletion is performed.
REALLY libraries with or without hg-rNONE depletion were deeply sequenced to ~20M reads and percent reads mapping to hemoglobin genes were analyzed. We observed robust depletion of highly expressed hemoglobin genes HBA1, HBA2, and HBB and other low abundance hemoglobin genes such as HBD, HBM, HBQ1
Correlation of unique genes expression (non ribosomal or hemoglobin) was evaluated between REALLY rNONE and REALLY hg-rNONE libraries (10 ng or 100 ng whole blood total RNA). Readcounts were generated with the STAR Aligner. Data filtering, normalization and log transformation was performed. We observed high pair-wise concordance between libraries (Pearson correlation shown) for both input amounts.
what about poor quality samples?
REALLY rNONE generates sequencing ready libraries from highly degraded FFPE RNA (RIN < 3) from as low as 10ng, whereas double stranded method are unable to generate libraries even after 16 cycles of PCR. The method does not require additional steps to modify or repair the input RNA unlike other methods. The resultant libraries have high mapping quality and duplication percent. High concordance is observed between low and higher inputs of degraded RNA.
Sequencing metrics and comparison between REALLY rNONE™ libraries and dsPrep libraries.
