Solid 3 plus system

Aliquots of 2 ml were centrifuged for sediment the cells, at 5000 g
and 4°C for 10 minutes. The bacterial total RNA was extracted using
ChargeSwitch® Total RNA Cell Kits(Invitrogen, USA) according to the manufacturer’s instructions, and
adding a mechanical lysis step using a Precellys^© grinder (Bertin Technologies, France). The total RNA
amount was measured by fluorometry using a Qubit™
- Quant-iT™ RNA Assay Kit (Invitrogen, USA). Messenger RNA
was enriched using Ribominus™ Transcriptome
Isolation Kit (Yeast and
Bacteria) (Invitrogen, USA), and the amount of recovered
RNA was measured as described above.
The enriched RNA samples were used to build strand-specific RNA-Seq
libraries. Two libraries, one at each experimental condition (0°C and
37°C, designated R1-0 and R1-37, respectively), were prepared using a
SOLiD™ Total RNA-Seq Kit. Fifty-base pair (bp) fragments were sequenced
using the SOLiD™ 3 Plus system according to the manufacturer’s
instructions (Applied Biosystems, USA). Two replicates from each
experimental condition (0°C and 37°C, designated as R2-0 and R3-0 and as
R2-37 and R3-37, respectively) were employed in the preparation of
fragment libraries using Ion Total RNA-Seq Kit and sequenced using 316
chip in an Ion Torrent Personal Genome Machine™ (Life Technologies, USA).
All the procedures were performed according to the manufacturer’s
instructions.

All INH-resistant isolates had previously been screened for known mutations found in katG, inhA, kasA, ndh genes, and the regulatory regions of inhA and ahpC, and had no detectable mutations [7] (link), [14] (link), [23] (link). Whole-genome sequencing was done in two stages. In the first stage, DNA from 12 INH-resistant and 6 INH-susceptible isolates was sequenced on the Applied Biosystems SOLiD 3 plus system by Mission Biotech Co., Ltd. Taiwan and AITBIOTECH Pte Ltd., Singapore. However, preliminary Sanger sequencing analysis showed that many of the mutations detected in INH-resistant isolates were also found in INH-susceptible isolates (data not shown). In the second stage, DNA from 11 additional INH-susceptible isolates were subjected to whole-genome sequencing to facilitate the selection of INH-resistant specific mutations, and these were sequenced using the Illumina HiSeq2000 Sequencing System by 1^st Base Pte Ltd, Singapore.