GSM4593626: Mutant Histone A rep 1; Thermococcus kodakarensis; RNA-Seq
Cultures were rapidly chilled and pelleted at 10,000 rpm for 5 min and then resuspended in 1.0 mL of Trizol (Invitrogen) with a 10-minute incubation at room temperature. 200 µl of chloroform was added followed by centrifugation at 15,000 rpm at 4 °C for 15 minutes yielding an RNA-containing aqueous layer which was added to 500 µl of isopropanol and incubated at room temperature for 10 minutes. Centrifugation at 15,000 rpm for 15 minutes at 4° C yielded an RNA pellet that was washed with 1 ml of 75% EtOH and subsequently resuspended in 88 µl of RNase-free H2O, 10 µl of DNaseI buffer and 1 µl of DNaseI (New England Biolabs) to digest residual DNA (37° C for 30 minutes). A second triplicate of TS600 and a triplicate of TS620 were prepared identically. TS600 and TS620: 1.5 µg of RNA was processed at Novogene for Prokaryotic RNA-seq, specifically for rRNA depletion (Ribo-ZeroTM Magnetic Kit), library construction (NEBNext® Ultra™ RNA Library Prep kit) and 150 bp paired-end sequencing on a HiSeq Illumina® platform. TS600 and TS622: Using ~120ng of RNA that was ribosomally depleted following the NEBNext® rRNA Depletion Kit (E6310), cDNA libraries were constructed with New England Biolabs NEBNext® Ultra™ Directional RNA Library Prep kit for Illumina® (E7420s) and NEBNext® Multiplex Oligos for Illumina® (Index primer set 1, E7335) according to the manufacture's procedure. The multiplexed libraries were sequenced at Cofactor Genomics using one high output NextSeq Illumina® run for single-end reads with a minimum read length of 75 bp and with a requested > 40 million reads per sample.
GEO
GSM4593626