title: GSM1563000: M-P 4h; Escherichia coli K-12; RNA-Seq
library_construction_protocol: RNA was isolated from the samples using RiboPureTM -Bacteria Kit (Ambion) following the manufacturer’s instructions. Once isolated, ~10g total RNA was treated with 8 units DNase (Invitrogen) twice to remove genomic DNA, and the complete removal of DNA was confirmed  by 35 cycles PCR amplification of a 196 bp fragment of the crp gene (5’-primer:AGCATATTTCGGCAATCCAG;3’-primer:TACAGCGTTTCCGCTTTTTC).  rRNAs were depleted from the total RNA using a MICROBExpress kit (Ambion) to enrich mRNAs. In our earlier experiments, sequencing was done on an Illumina GAII platform at the sequencing core facility of the University of North Carolina at Chapel Hill, and the directional RNA-seq libraries were constructed using Illumina Small RNA Sample Prep Kit following the vendor’s instruction with some modifications. Briefly, after the purified mRNA was fragmented using a RNA fragmentation kit (Ambion), the fragmented RNA was treated with Antarctic phosphatase (NEB) to remove the 5’-tri-phosphate groups of RNAs with an intact 5’-end. A mono-phosphate group was then added back to the 5’-end of RNAs by polynucleotide kinase (PNK, NEB) in the presence of 10 mM ATP.  The v1.5 sRNA 3’ Adaptor  (5'/5rApp/ ATCTCGTATGCCGTCTTCTGCTTG /3ddC/) was ligated to the 3’-end of fragmented RNAs using truncated T4 ligase 2 (NEB), and the SRA 5’ RNA adaptor (5'GUUCAGAGUUCUACAGUCCGACGAUC) was ligated to the 5’-end of fragmented RNAs using T4 ligase. To preserve short inserts from small RNAs we omitted the size selection step after PCR application of the inserted RNA fragments. For our later experiments, sequencing was done on an Illumina HiSeq 2000 platform at David H. Murdock Research Institute of the North Carolina Research Campus (Kannapolis, NC), and we constructed the directional RNA-seq libraries using Illumina’s TruSeq Small RNA Sample Prep Kit, so that multiplex sequencing can be achieved by using the barcoded PCR primers. The details of the method will be described elsewhere (Dong, Li and Su).  Briefly, after similar treatments as described above, the 5’ Adapter (RA5: 5’ GUUCAGAGUUCUACAGUCCGACGAUC), and 3’ Adapter (RA3: 5’ TGGAATTCTCGGGTGCCAAGG) were ligated to 5’- and 3’-end of fragmented RNAs, respectively. Reverse transcription-PCR (RT-PCR) was performed using SuperScript II Reverse Transcriptase Kit (Invitrogen) using the SRA RT primer, followed by 16 cycles of PCR amplification. Again, the size selection was omitted on PCR products to preserve short inserts from possible small RNAs. Single-end sequencing on the Illumina GA II platform was done with 76 cycles, while that on the HiSeq 2000 platform was done with 100 cycles. Libraries for some samples (M-C1h and M-C2h) were prepared by the two methods and sequenced on the two different platforms.
attributes.tag: GEO Accession
attributes.value: GSM1563000