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.

With rNONE™, our streptavidin-based workflow for ribodepletion, we chose to perform removal 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.

Finally our REALLYrun open-source software, takes away the guesswork from RNA-Seq data-analysis. Visit our software page to get download instructions.

Product Specifications

Reduced protocol time by more than 3 hours in comparison to typical RNA-Seq protocols

Do not need additional modules to retrieve degraded samples
Input Range 10-250 ng
NEW! 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- rNONE™ kit components

First-strand cDNA synthesis module
SRSLY® Library Preparation Base Kit for adapter ligation and Index PCR
rNONE™ - Ribodepletion module with rNONE purification beads
Clarefy™ DNA purification beads
Visit our kit component page for more information

REALLY FAQS

Order here

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

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™ PERFORMANCE METRICS

**Comparative molecular metrics of REALLY 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 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.

**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.** 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

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.