Description
Material provided: data
Unit definition:
Library Preparation: SMARTer smRNA
44 million reads for 1×50 Single Read Length (suggested at least 3.5 million reads per sample – 12 samples per run).
Short Description: Small noncoding RNAs act in gene silencing and post-transcriptional regulation of gene expression. Small RNA sequencing (RNA-Seq) is a technique to isolate and sequence small RNA species: miRNAs, siRNAs, piRNAs, and snoRNAs with a size range of 20 to 30 nucleotides, that play a crucial role in regulating gene expression.
Ideal for the discovery and the profiling of all forms of small non-coding RNAs and to examine the differential expression of all small RNAs in a given sample.
Benefits of generating small RNA sequencing libraries directly from total RNA to understand the role of noncoding RNA:
- Understanding how post-transcriptional regulation contributes to phenotype
- Identification of novel biomarkers
- Capture of complete range of small RNA and miRNA species
The experiment and the product
The service for Small RNA Illumina sequencing + bioinformatics analysis support (Ref-SKU: S.1.4.IT.149.43) includes:
– Library preparation with SMARTer smRNA – seq chemicals provided by Takara including the following for each sample:
- 3’ Polyadenylations
- First strand synthesis and tailing by RT
- Template switching and extension by RT
- Addition of full length Illumina adapters by PCR
- Library Validation
- Library Normalization for cluster generation
- Sequencing: 1 x 50 SR using MiSeq Reagent Kit v3
Agilent Tape Station 4200 trace for the small RNA library validation step positive control
– Bioinformatics analysis service (Ref-SKU: S.1.4.IT.149.47) includes the following for each sample:
- Quality control and pre-processing of raw data;
- Alignment of reads to the reference genome, count and dereplication of small RNA sequences;
- Known miRNA identification by sequence homology with miRBase repository.
End-user responsibilities
Biological material to be provided by the end-users and described in the dedicated request form.
RNA INPUT RECOMMENDATIONS
For the small RNA library preparation, the Polo d’Innovazione Genomica Genetica Biologia, follows the library preparation protocol from Illumina. The kit protocol has been optimized for:
– option 1) Total RNA Input: a minimum of 100ng of total RNA in 20μl nuclease free water with an absorbance ratio values of 1.8 – 2.0.
– option 2) Purified Small RNA Input: a minimum of 1050 ng of purified small RNA in 20μl nuclease free water with an absorbance ratio values of 1.8 – 2.0.
Recommended RNA Integrity Number (RIN) value: ≥ 8. Please make sure that your RNA is free of contaminants (DNA and proteins) and has been extracted using a method that preserves small RNA fractions.
– REMARKS:
- If the provided sample doesn’t comply with the above requested specifications for “RNA INPUT RECOMMENDATIONS”, the customer will be contacted for a re-sampling.
- In case of failure of the sequencing run, the service will be repeated.
– DESPATCH OF SAMPLES
- Safe-lock 1.5 – 2 ml tubes properly capped to be used to avoid accidental evaporation or sample contamination during shipping.
- Samples to be labelled correctly.
- Correct temperature must be ensured during the shipment to avoid sample degradation.
- Complete the enclosed Sample Submission form and return together with the samples to: orders@pologgb.com.
Supplier responsibilities
– STORAGE OF BIOLOGICAL SAMPLE
Polo d’Innovazione Genomica Genetica Biologia will store the analysed samples only for 1 month after delivery. Extended storage time is available for a maximum of 4 months and must be requested in advance of the experiments in the service request application.
– STORAGE OF DATA
Polo d’Innovazione Genomica Genetica Biologia waives any responsibility for the storage and backup of the supplied project and sequencing data after delivery to the end-user. Polo d’Innovazione Genomica Genetica Biologia will store the project data only for 1 month after delivery. After expiration of this period, all data are removed from our servers and archived (unless otherwise agreed with the end-user). Your data might be re-imported at an extra cost within the period of 1 year after delivery of the final data. After 1 year, all project data will be deleted permanently from our systems. Extended data storage is available on request at an extra cost and must be requested at project start.
– DELIVERY OF RESULTS
The bioinformatics results will be sent according to the Modalities of Transfer with a Bioinformatic Reports PDF Files. The bioinformatic analysis will be executed in 45 days from the generated sequencing data.
Modalities of transfer:
- by “WeTransfer” platform to email for data of up to 2Gb
- by “MegaSync” platform for data ranging from 2Gb to 15Gb
- by HD for data over 15 Gb
Please note that the cost of HD driver is not included. Where requested, the end-user must provide the HD.
For further details on the description of the experiment please consult the “SMARTer® smRNA-Seq Kit for Illumina® User Manual”
For more information, please contact us.
For technical assistance, contact Polo GGB Technical Support:
Email: techsupport@pologgb.com
Telephone: 0039 0577 381310