RNA-Seq Library Preparation with rRNA Depletion

We prepared a library using 1 μg K-562 total RNA plus 2 μl of 1:100 diluted ERCC spike-in mix 1 (Ambion) using the TruSeq RNA-Seq kit (Illumina) with the following modifications. (1) The rRNA was depleted using the RNase H method^{19 (link)} instead of using oligo (dT) selection. (2) We eluted the rRNA depleted RNA from the SPRI beads using EPF buffer from the TruSeq kit and heated at 70°C for 10 minutes. (3) We used a different set of barcoding indices rather than those in the TruSeq kit for the ligation and final PCR steps.

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Adiconis X., Haber A., Simmons S.K., Moonshine A.L., Ji Z., Busby M.A., Shi X., Jacques J., Lancaster M.A., Pan J.Q., Regev A, & Levin J.Z. (2018). Comprehensive comparative analysis of 5’ end RNA sequencing methods. Nature methods, 15(7), 505-511.

Publication 2018

ERCC spike-in mix 1 TruSeq RNA-Seq kit

Buffer Library Ligation Oligo Rna seq Rnase h Rrna

Corresponding Organization :

Other organizations : Broad Institute, MRC Laboratory of Molecular Biology, Medical Research Council

Top 5 similar protocols

Variable analysis

independent variables

(1) The rRNA was depleted using the RNase H method instead of using oligo (dT) selection.
(2) We eluted the rRNA depleted RNA from the SPRI beads using EPF buffer from the TruSeq kit and heated at 70°C for 10 minutes.
(3) We used a different set of barcoding indices rather than those in the TruSeq kit for the ligation and final PCR steps.

dependent variables

Not explicitly mentioned.

control variables

1 μg K-562 total RNA
2 μl of 1:100 diluted ERCC spike-in mix 1 (Ambion)
TruSeq RNA-Seq kit (Illumina)

positive controls

ERCC spike-in mix 1 (Ambion)

negative controls

Not explicitly mentioned.

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!