Whole Cell Ribosome Extraction and RNA-seq

Whole cell extracts were prepared from 2 × 10⁹ cells as described (30 (link)); after extraction with 0.3M KCl, the ribosomes were removed by centrifugation for 2 h at 33 000 rpm in a Beckman 70.1Ti rotor (150 000 × g). RNA extracted from the post-ribosomal supernatant (PRS) was used for library preparation, essentially as described (45 (link)) and size selected on a E-Gel EX (Thermo Scientific Ltd). The libraries were sequenced on an Illumina NextSeq machine.

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Chikne V., Shanmugha Rajan K., Shalev-Benami M., Decker K., Cohen-Chalamish S., Madmoni H., Biswas V.K., Kumar Gupta S., Doniger T., Unger R., Tschudi C., Ullu E, & Michaeli S. (2019). Small nucleolar RNAs controlling rRNA processing in Trypanosoma brucei. Nucleic Acids Research, 47(5), 2609-2629.

Publication 2019

70.1Ti rotor E-Gel EX NextSeq machine

Cell extracts Cells Centrifugation Library Ribosomal

Corresponding Organization : Bar-Ilan University

Other organizations : Weizmann Institute of Science, Yale University

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Variable analysis

independent variables

Whole cell extracts were prepared from 2 × 10^9 cells

dependent variables

RNA extracted from the post-ribosomal supernatant (PRS) was used for library preparation
The libraries were sequenced on an Illumina NextSeq machine

control variables

Whole cell extracts were prepared as described (30 (link))
After extraction with 0.3M KCl, the ribosomes were removed by centrifugation for 2 h at 33 000 rpm in a Beckman 70.1Ti rotor (150 000 × g)
The libraries were size selected on a E-Gel EX (Thermo Scientific Ltd)

positive controls

None specified

negative controls

None specified

Annotations

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