GSM3334093
GSM3334093: Term2; Streptomyces avermitilis; OTHER
Harvested cells were washed with polysome buffer (20 mM Tris-HCl pH 7.5, 140 mM NaCl, 5 mM MgCl2), and then resuspended with lysis buffer (0.3 M sodium acetate pH 5.2, 10 mM EDTA, 1% Triton X-100). The cell suspension was then frozen with liquid nitrogen, and lysed by grinding using mortar and pestle. The cell lysate was centrifuged at 4 oC for 10 minutes at 16,000 g and the supernatant was stored at -80 oC until used for RNA extraction. RNA was extracted by mixing with equal volume of phenol:chloroform:isoamyl alcohol = 25:24:1 solution. The mixture was then centrifuged and upper aqueous phase was recovered. The RNA was purified with ethanol precipitation. The purified RNA was treated with DNase I (New England Biolabs) to remove any genomic DNA contamination. Ribosomal RNA was depleted with Ribo-Zero rRNA Removal Kit Bacteria (Epicentre) according to the manufacturer's instructions. 500~900 ng of the rRNA-depleted RNA was mixed with 1 mL of 150 mM amino-blocked DNA adaptor (5'-p-NNAGATCGGAAGAGCGTCGTGT-3'), 2.5 uL of 10x T4 RNA ligase 1 buffer, 2.5 uL of 10 mM ATP, 2 uL of DMSO, 9.5 uL of 50% PEG8000, and 2.5 uL of T4 RNA ligase 1 (New England BioLabs). The mixture was incubated at 23 oC for 2.5 hours and purified with Agencourt AMPure XP beads (Beckman Coulter) and eluted with 9 uL DEPC-treated water. Then the RNA-adaptor ligates were fragmented using fragmentation buffer (Ambion) by incubating at 72 oC for 90 seconds. After fragmentation, the product was purified with Agencourt AMPure XP beads and eluted with 8 uL DEPC-treated water. The fragmented RNA was reverse transcribed using 1 uL of 10 uM reverse transcription primer (5'-TCTACACTCTTTCCCTACACGACGCTCTTC-3') with SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. After reverse transcription, the cDNA was purified with Agencourt AMPure XP beads and eluted with 5 uL DEPC-treated water. The purified cDNA was subjected to another adaptor ligation as above, with increased incubation time (8 hours) and different amino-blocked adaptor sequence (5'-p-NNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3'). After adaptor ligation, the product was purified using Agencourt AMPure XP beads and indexed by PCR for 10 cycles with Phusion High-Fidelity DNA Polymerase using forward (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCT-3') and reverse (5'-CAAGCAGAAGACGGCATACGAGATNNNNNN (6 nt index) GTGACTGGAGTTCAGAC-3') primers.
GEO
GSM3334093
GEO Accession
GSM3334093