GSM1251761: H3V single knockout parasite sample; Leishmania major; RNA-Seq
Logarithmically-growing promastigotes from WT L. major FV1, one H3V/HYG transfectant, and two Δh3v transfectants were collected and resuspended at a concentration of 5x108 cells/mL in TriZOL (Invitrogen). The aqueous phase was isolated by adding 0.2 mL chloroform (Fisher Scientific) and centrifugation at 12,000 x g for 15 minutes at 4°C. The aqueous phase was isolated and RNA was precipitated by adding 1 volume of 100% isopropanol (Fisher Scientific) and centrifugation at 12,000 x g for 10 minutes at 4°C. The RNA pellet was washed with 75% ethanol (Pharmco) and was resuspended in nuclease-free water (Ambion). Purified RNA was treated with 20 units of DNase I (Ambion) and was precipitated using 1/10 volumes 3M sodium acetate (Sigma) and 3 volumes 100% ethanol. RNA was pelleted by centrifugation at 15,000 g-1 and the resulting pellet was washed with cold 75% ethanol and resuspended in RNAse-free water (Ambion). Next Generation Sequencing (NGS) libraries were constructed by priming ~1 µg of total RNA from either the purine-starved or purine-replete sample with a random hexamer primer (TCCGATCTCTNNNNNNN) for first strand cDNA synthesis and an oligonucleotide primer (TCAGTTTCTGTA) derived from the Leishmania splice leader (SL) sequence for second-strand cDNA synthesis.  The resultant cDNA was amplified by PCR for 12-15 cycles using the oligonucleotides that overlapped with the initial primers and containing adapters for subsequent cluster generation and sequencing using Solexa sequencing by synthesis (SBS) technology.
GEO Accession
GSM1251761