title: GSM9102736: Study antibiotic 80998879 sample 6; Mycobacterium avium; RNA-Seq
library_construction_protocol: Bacteria cultures of 10 ml were centrifuged in Falcon tubes and subsequently resuspended in 0.5 ml TRIzol reagent (Invitrogen) and transferred to 2 ml Eppendorf tubes containing 0.25 ml glass beads (0.1 mm, Qiagen).  Cell lysis was performed by bead-beating in a bullet blender (Next Advance), for 1 min  at highest speed, for 6 cycles. The tubes were suspended in ice for 2 min between cycles. After 5 minutes centrifugation at 20.238 RCF (at RT), the TRIzol layer was recovered and  chloroform added (1:5) and vortexed vigorously for 15 seconds. Samples were centrifuged for 15 min at 21.130 RCF (at 4° C), upper transparent aqueous layer was carefully removed and mixed with 0.3 ml isopropanol and spun down 10 minutes. Pellets were washed with 0.5 ml 70% ethanol and centrifuged for 15 minutes at 21.130 RCF (at 4 ° C). Ethanol was removed as much as possible and subsequently air dried  for 5 minutes and 20 microliter RNAse free water was added. Equipment: Centrifuge Eppendorf 5424 R (cooled max 21130 RCF) or 5424 (max 20.238 RCF) The library and sequencing was performed by the company Novogene (UK). Firstly, ribosomal RNA was removed from total RNA, followed by ethanol precipitation. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. During the second strand cDNA synthesis, dUTPs were replaced with dTTPs in the reaction buffer. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. Following is the workflow of library construction: The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries have been pooled and sequenced on Illumina platforms, according to effective library concentration and data amount specified.