title: GSM1621965: DL3; Mycobacterium tuberculosis; RNA-Seq
library_construction_protocol: RNA was isolated using phenol-chloroform extraction and cell disruption with BeadBeater (BioSpec Products, Bartlesville, OK). RNA was treated with Turbo DNase (Life Technologies) to remove traces of genomic DNA, and purified with the RNeasy mini kit (Qiagen, Venlo, Netherlands). RNA samples were depleted of 16S and 23S rRNA with MicrobExpress kit (Life Technologies) (Samples Log 1-3, DM 1-3 and DE 1) or Ribo-Zero (Epicentre) (Samples DE 2,3 and DL 1-3). cDNA for Illumina sequencing was prepared according to “Directional mRNA-Seq Sample Preparation” guide (Part # 15018460 Rev. A) issued by Illumina: RNA was sheared with RNA Fragmentation Reagents (Ambion) for 10 minutes, and ethanol precipitated. Fragmented RNA was dephosphorilated with Antarctic phosphatase (NEB) and 5' phosphate was added with T4 Polynucleotide Kinase (NEB). RNA was purified with RNeasy MinElute kit according to modified protocol from "Directional mRNA-Seq Sample Preparation" guide. Illumina 3' and 5' RNA adapters were ligated according to "TruSeq™ Small RNA Sample Preparation Guide". RNA was reverse transcribed and amplified for 11 cycles. PCR products were fractionated on PAGE and those with the lengths of cDNA inserts 30-80 nt were excized and eluted from the gel. These fragments were sequenced by running 77 cycles on Illumina HiSeq 2000 sequencer.
attributes.tag: GEO Accession
attributes.value: GSM1621965