GSM2493799: BIP1; Streptococcus pneumoniae TIGR4; RNA-Seq
For HIP-RNA, hearts were excised from HIP-infected mice (n=3) when deemed moribund. Hearts were rinsed, diced, fragments washed with ice cold PBS, and the fragments homogenized in RNAprotect bacteria reagent (Qiagen). For BIP-RNA, 10 mice pre-depleted for neutrophils using anti-Ly6G antibody (BioXCell, clone RB6-8C5), were infected with TIGR4 and blood was collected in RNAprotect bacteria reagent (Qiagen) when the mice were deemed moribund such that blood from 5 mice were pooled as one sample (n=2 BIP samples). These heart homogenates were stored at -80°C. On the day of total RNA isolation from heart homogenates, the samples were thawed and spun down to discard the supernatant. The pellets were further homogenized in 600 μL RLT with B-ME buffer using a motorized mortar for 30 seconds. The re-homogenized samples were then disaggregated in a Qiashredder followed by RNA extraction with the RNeasy Micro Kit (Qiagen) with DNase treatment on column and in solution. The isolated RNA was quantitated using Nanodrop and Bioanalyzer. Samples were then depleted of rRNAs using the Ribo-Zero rRNA Removal Kit for Gram-positive bacteria and human/mouse/rat (Illumina, San Diego, CA). For the in vitro biofilm and planktonic samples: planktonic mid-log phase (OD620nm = 0.5) TIGR4 grown in THB were used to seed continuous once-flow through biofilm reactors. Biofilms were allowed to grow for 48 hours prior to collection of bacteria. RNA was isolated from the planktonic seed cultures and their respective biofilms. In total, 3 independent biological samples were collected with planktonic and biofilm RNAs from each experiment paired. Total RNA was extracted from each replicate separately using enzymatic lysis of pneumococcal cells (10 μL mutanolysin, 20 μL proteinase K, 15 μL lysozyme, and 55 μL TE) followed by RNA extraction using the same protocol mentioned above. Blood samples were isolated in a similar manner as biofilm and planktonic samples, including enzymatic lysis, except that cells were first disaggregated with the Qiashredder like for the heart samples. RNA libraries were prepared for sequencing using standard Illumina protocols