SLAM Sequencing

Thiol (SH)-linked alkylation for the metabolic sequencing of RNA, short for SLAM-Seq, is an orthogonal-chemistry-based transcript-mapping method that detects 4-thiouridine (s4U) (a type of nucleoside analog) which incorporates into total RNA at single-nucleotide resolution. SLAM-Seq can be used to map the dynamics of RNA-polymerase-II-dependent gene expression and is an accessible, rapid, scalable, and cost-effective method for studying mechanisms of gene regulation.

Overview

SLAM-Seq is a next-generation sequencing (NGS)-based technology for direct quantification of 4-thiouridine (s4U)–labeled mRNAs within the total mRNA pool. This procedure is achieved through the introduction of s4U to thymine and alkylation of the thiol group, which makes modified T deem to be cytosine and induces misincorporation of guanine during reverse transcription instead of adenine, enabling the detection of s4U as thymine-to-cytosine conversion in 3' -end mRNA-sequencing. By sequencing these G mismatches, the newly synthesized mRNA, or other long RNAs and small RNAs can be directly quantified. Integrating rapid chemical-genetic perturbation and SLAM-seq establishes a simple yet powerful strategy for probing specific and global direct functions of transcription factors and cofactors. Meanwhile, SLAM-seq also allows for the study of intracellular RNA dynamics, from transcription, RNA processing to RNA stability when combined with metabolic labeling protocols.

Features

High Sensitivity High Specificity Bioinformatics Analysis One-stop Service
High resolution and sensitivity Long RNA probe length provides high binding affinity to the target lncRNA Our integrated bioinformatics pipeline can be tailored to suit your project. Provides one-stop service for library construction, sequencing, sample QC and data analysis.

Project Workflow

Sample Preparation

1. Sample Preparation

RNA purification;
quality assessment and quantification.

Library Preparation

2. Library Preparation

RNA fragmentation;
cDNA library preparation.

Sequencing

3. Sequencing

Illumina HiSeq;
PE 50/75/100/150.

Data Analysis

4. Data Analysis

Visualize and preprocess results, and perform custom bioinformatics analysis.

SLAM Sequencing

Bioinformatics Analysis Pipeline

In-depth data analysis:

  • Data quality control;
  • Reference-based mapping;
  • SNP calling;
  • Gene annotation;
  • Thymine-to-cytosine (T>C) counts and expression quantization (longRNA);
  • Total T coverage;
  • Two-sided beta-binomial test;
  • GO and KEGG pathway analysis

Sample Requirements

RNA sample (concentration ≥ 1 ng/µL, quantity ≥ 5 µg)
1.8 ≤ OD260/280 ≤ 2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0.
Please make sure that the RNA is not degraded nor contaminated.

Sample storage: RNA can be dissolved in ethanol or RNA-free ultra-pure water and stored at -80°C. RNA should avoid repeated freezing and thawing.

Shipping Method: When shipping RNA samples, the RNA sample is stored in a 1.5 mL Eppendorf tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable: FastQ, BAM, coverage summary, QC report, full statistical analysis & alignments, custom analysis reports on customer request.

References:

  1. Muhar M, Ebert A, Neumann T, et al. SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis. Science. 2018, 360(6390): 800-5.
  2. Matsushima W, Herzog VA, Neumann T, et al. SLAM-ITseq: sequencing cell type-specific transcriptomes without cell sorting. Development. 2018, 145(13): dev164640.
  3. Herzog VA, Reichholf B, Neumann T, et al. Thiol-linked alkylation of RNA to assess expression dynamics. Nature Methods. 2017, 14(12): 1198-204.
* For Research Use Only. Not for use in diagnostic procedures.


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