alias: GSM2046125
title: GSM2046125: BG880 rep. 2; Bacillus subtilis; RNA-Seq
library_construction_protocol: Total RNA was extracted with hot phenol method, and ribosomal RNA was removed with Ribo-Zero rRNA Removal Kit for bacteria For regular RNA-seq, Libraries were constructed according to the standard Illumina RNA-seq protocol. For PARE, equal amounts of samples were used to construct the PARE libraries using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set 1). The manufacturer’s protocol was modified so that 5ʹ monophosphorylated ends that result from endonuclease cleavage or pyrophosphohydrolase activity were preferentially selected. Briefly, after 3ʹ SR Adaptor ligation, reverse transcriptase (RT) primer hybridization, and 5ʹ SR Adaptor ligation – performed as per the manufacturer’s protocol – the reaction mixture volume was brought to 100 μl and samples were cleaned up with the RNeasy MinElute Cleanup Kit (Qiagen). 12 μl of RNA was recovered following the manufacturer’s protocol. The RNA was fragmented using the NEBNext® Magnesium RNA Fragmentation Module at 94°C for 4 minutes, and 2 μl of stop solution was added. After a second RNeasy MinElute cleanup, half of the RNA (6 μl) was again ligated to the 3ʹ SR Adaptor. Thus, after magnesium fragmentation only RNAs that retained their 5ʹ SR Adaptor (and original 5’ monophosphate) would undergo subsequent RT and PCR amplification. After addition of the RT primer, the reaction volume was adjusted to 30 μl and RT/PCR amplification reactions were performed as per the manufacturer’s protocol, using barcoded primers. One tenth of the PCR reaction was run on a 1.5% agarose gel to visualize the library, which consisted of cDNA ranging from 50 to ~500 nts. 20 μl of the cDNA library were prepared for Illumina sequencing using a 1.5X volume of Agencourt AMPure XP Beads (Beckman Coulter), according to the manufacturer’s instructions. The procedure was repeated twice, and the sample was eluted in a final volume of 20 µl.
identifiers.namespace: GEO
identifiers.id: GSM2046125