GSM9364076: WhiB1 experiment TSS noWhiB1 rep1 (RNAP-SigA-RbpA-CarD); Mycobacterium tuberculosis; RNA-Seq
Genomic DNA was extracted and prepared from Mtb (wildtype H37Rv) using the CTAB-lysozyme method with phenol-chloroform-isoamyl alcohol extraction. RNA was isolated from in vitro transcription reactions using the Zymo Research RNA Clean & Concentrator-5 column purification kit. 5' end libraries were prepared using the following procedure: blocking the 3' ends by ligating a 3'SpC3 ssDNA adaptor; converting 5'PPP to 5'P; ligating a 5' ssDNA adaptor with replicate-specific inline barcodes; pooling samples; fragmenting RNA to Illumina-appropriate size using zinc; repairing transcript ends; degrading non-5' end RNA fragments; ligating a 3' ssDNA adaptor complementary to a specific reverse transcription primer; carrying out reverse transcription and digesting RNA in the RNA-DNA hybrids with RNase H; and amplifying the final library using primers and indices from the NEBNext Small RNA Library Prep Kit. 3' end libraries were prepared using the following procedure: ligating a 3'SpC3 ssDNA adaptor with replicate-specific inline barcodes and reverse transcription primer handle; pooling samples; fragmenting RNA to Illumina-appropriate size using zinc; carrying out reverse transcription and digesting RNA in the RNA-DNA hybrids with RNase H; and amplifying the final library using primers and indices from the NEBNext Small RNA Library Prep Kit. Mtb genomic DNA samples were prepared by the Rockefeller University Genomics Resource Center using their full service library preparation and sequencing option for whole-genome sequencing.