title: NextSeq 500 sequencing; Essential role of proline synthesis and the one-carbon metabolism pathways for systemic virulence of Streptococcus pneumoniae
design: Essential role of proline synthesis and the one-carbon metabolism pathways for systemic virulence of Streptococcus pneumoniae
library_construction_protocol: Triplicate cultures of each S. pneumoniae strains were grown in THY broth until an OD595 0.4-0.5, then centrifuged, resuspended in RNAprotect stabilization buffer (Qiagen), centrifuged again before RNA extraction using Mirvana RNA kit (Applied biosystems) according to the manufacturer's 485 instructions with an additional lysis step using vigoruous shaking of the tubes after addition of 0.1 mm glass beads (MP Biomedicals). For the serum studies, bacteria were grown in THY broth up to OD595 0.4-0.5, washed twice in PBS and resuspended in 500 ul of fresh human sera for 60 minutes NA Triplicate cultures of each S. pneumoniae strains were grown in THY broth until an OD595 0.4-0.5, then centrifuged, resuspended in RNAprotect stabilization buffer (Qiagen), centrifuged again before RNA extraction using Mirvana RNA kit (Applied biosystems) according to the manufacturer's 485 instructions with an additional lysis step using vigoruous shaking of the tubes after addition of 0.1 mm glass beads (MP Biomedicals).  For the serum studies, bacteria were grown in THY broth up to OD595 0.4-0.5, washed twice in PBS and resuspended in 500 ul of fresh pooled human sera collected from healthy volunteers for 60 minutes following the same protocol mentioned above before RNA extraction.After 60 minutes incubation bacteria were centrifuged, resuspended in RNAprotect stabilization buffer (Qiagen), centrifuged again before RNA extraction using Mirvana RNA kit (Applied biosystems) according to the manufacturer's 485 instructions with an additional lysis step using vigoruous shaking of the tubes after addition of 0.1 mm glass beads (MP Biomedicals) NA RNA for RNAseq were extracted from double mutant strains cultured to an OD600 0.4-0.5 using Mirvana RNA kit (Applied biosystems) with an additional physical lysis step using 0.1 mm glass beads (MP Biomedicals), treated with Turbo DNAse (Applied biosystems), and deleted of ribosomal RNA using Ribo-Zero Magnetic Kit Bacteria (Illumina) before preparation of sequencing libraries Sequencing libraries were prepared using the KAPA RNA HyperPrep kit (Roche Diagnostics) and 8 cycles of amplificationKAPA RNA HyperPrep Kit - Automated Library Prep. The process starts with  RNA fragmentation and priming, first strand cDNA synthesis, second strand cDNA synthesis and A-Tailing, adapter ligation, two post-ligation cleanups, and is followed by an off-deck PCR amplification. This module can be completed in approximately 4 hours