HEK293T cells in a 6-well format were transfected with 1-ug of an individual cshRNA vector. Forty-eight hours post-transfection, small RNAs (<70 nts) were isolated from total RNA as described in (20 (link)) and pooled. Small RNA libraries were prepared for Illumina small RNA sequencing from the pooled small RNAs using the multiplex small RNA library prep set (New England Bio Labs) and sequenced on a Illumina HiSeq 2500 and NextSeq 500 platforms (GSAF, UT Austin). Adapter sequences were trimmed from the reads using fastx clipper from the FASTX-Toolkit software (http://hannonlab.cshl.edu/fastx_toolkit ). The preprocessed reads were then mapped to cshRNA reference sequences using SHRiMP2 software package (23 (link)). Small RNA reads were quantitated using custom scripts.
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Genome-wide cshRNA screening by small RNA-seq
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Corresponding Organization : The University of Texas at Austin
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- Transfection of HEK293T cells with 1-ug of an individual cshRNA vector
- Small RNA reads quantitated using custom scripts
- HEK293T cells in a 6-well format
- Small RNAs (<70 nts) isolated from total RNA as described in (20)
- Small RNA libraries prepared for Illumina small RNA sequencing from the pooled small RNAs using the multiplex small RNA library prep set (New England Bio Labs)
- Sequenced on a Illumina HiSeq 2500 and NextSeq 500 platforms (GSAF, UT Austin)
- Adapter sequences trimmed from the reads using fastx clipper from the FASTX-Toolkit software
- Preprocessed reads mapped to cshRNA reference sequences using SHRiMP2 software package (23)
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