Usage Instructions:

1. This kit provides a complete ribosome profiling (Ribo-seq) solution, including reagents required for ribosome-protected fragment (RPFs) extraction, RNA fragmentation, library construction and purification, suitable for translatomics research.

2. Use fresh or liquid nitrogen flash-frozen cells (≥1×10^6 cells). After cell lysis, centrifuge to collect the supernatant. It is recommended to add cycloheximide (final concentration 100 μg/mL) to stabilize ribosomes.

3. Add 10 μL of RNase I (provided in the kit), incubate at 25°C for 45 minutes. Purify RPFs using the spin column provided in the kit, elute with 30 μL.

Frequently Asked Questions:

Q1: Possible reasons for low RPF yield?

Incomplete cell lysis: Optimize lysis buffer ratio or extend lysis time.

Low RNase I activity: Check enzyme storage conditions (avoid repeated freeze-thaw cycles) or extend digestion time.

Q2: How to resolve high rRNA contamination in sequencing data?

It is recommended to use an rRNA removal kit (e.g., Ribo-Zero) before library construction.

Check RNase I digestion efficiency to ensure ribosomes are not over-degraded.

Q3: Abnormal library fragment size (not 28 nt)?

Possible due to excessive RNA fragmentation: Shorten the reaction time at 70°C to 5–8 minutes.

Check T4 PNK enzyme activity to ensure complete end repair.

Q4: Which sequencing platforms are compatible with this kit?

Default adapter design is compatible with Illumina platforms (supports PE50/PE100). Adapters for other platforms can be customized upon request.

References:

Ingolia, N.T. et al. (2009). Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science, 324(5924), 218–223.

Kit Optimization Guide: *[Attachment] RB-1000_Optimization_Guide.pdf*