GSM5172776: pCF10_P23IGR_pCIE_U_FLAG2; Enterococcus faecalis; RNA-Seq
Cell lysates were prepared by suspending cells in 500 µL of TBS buffer with 5 µL of HALT protease inhibitor cocktail and 2 µL of superase.  Cells were added to a pre-chilled tube containing 500 µL of glass beads (0.1 mm) and disrupted by repeated cycles of maceration for 40 sec at 400 rpm with intervals on ice. TBS buffer (500 µL) was added and the slurry was vortexed for 30 sec, after which cell fragments and other material were pelleted by centrifugation 12,000 x g for 30 min at 4 °C. The supernatant was transferred to a pre-chilled tube, the volume was increased to 1 mL with cold TBS buffer, and 5 µL of superase inhibitor was added. An aliquot representing the input fraction was removed, and the remaining material was subjected to FLAG pull-downs by addition of 75 µL of anti-FLAG magnetic bead slurry to a 2.0 mL dolphin tube (Costar 3213).  The mixture was then placed on a nutator (rocker) for 2.0 h at 4 °C, after which time the resin was pelleted by centrifugation at 4,000 x g for 30 sec at 4 °C.  The supernatant was removed, and the resin washed 3 x with 1.5 mL of TBS buffer, with incubations on a nutator for 1 min and then subjected to magnetic bead pull-downs at 4 °C.  Material bound to the FLAG beads was eluted by addition of 15 µL 5 µg/µL 3xFLAG peptide solution and 250 µL of 3xFLAG elution buffer.  Ribosomal RNA was depleted using a MicrobeExpress kit.  All RNA samples were treated with Turbo DNase (ThermoFisher, rigorous method described by the manufacturer) and submitted to the University of Minnesota Genomics Center for Illumina library prep. Pull-down samples were processed using the Illumina TruSeq stranded mRNA preparation kit. To retain small RNAs, size selection was not performed between library preparation and sequencing for pull-down samples. All samples were sequenced as paired-end reads on an Illumina HiSeq 2500 in high output mode (125 bp read length for whole-cell samples and 50 bp read length for pull-down samples). Sequencing files were trimmed to remove contaminating adapter sequences and low-quality bases using Trimmomatic.  Reads were mapped to the pCF10