GSM2837323: LF82 transcripts (without DSS, Mouse#11); Escherichia coli LF82; RNA-Seq
Total RNA was extracted by combining Phenol:Chloroform:Isoamyl Alcohol (pH4.5, Ambion 9720) and LiCl precipitation (Ambion) according to the manufacturer's instructions. Then, isolated RNAs were treated with DNase (Ambion) and RNeasy (QIAGEN). Further, bacterial ribosomal RNA was depleted by Ribo-zero kit (illumina). Isolated RNA was assessed for quality using the TapeStation (Agilent). Samples with RNA Integrity Numbers (RINs) of 8 or greater were prepped using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB), NEBNext Ultra RNA Library Prep kit (NEB) and NEB Next Multiplex Adaptors for Illumina (NEB, E7335, E7500, E6609, E7600). Where (10ng up to 1μg) of total RNA was converted to mRNA using a poly A purification. The mRNA is then fragmented and copied into first strand cDNA using reverse transcriptase and random primers. The 3 prime ends of the cDNA were then adenylated and adapters were ligated. One of the adapters that was ligated has a 6 nucleotide barcode that will be unique for each sample which allowed us to sequence more than one sample in each lane of the Illumina HiSeq flow cell. The products were purified and enriched by PCR to create the final cDNA library. Final libraries were checked for quality and quantity by TapeStation (Agilent) and qPCR using Kapa's library quantification kit for Illumina Sequencing platforms (Kapa Biosystems). Sixteen samples were multiplexed and pooled. They were clustered on the cBot (Illumina) and sequenced over two lanes on a 50 cycle single end on a HiSeq 2500 in High Output mode using version 4 reagents according to manufacturer's protocols
GEO Accession
GSM2837323