GSM4767238: PinT_in_vivo_rep2; Mus musculus; Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344; RNA-Seq
Eluted RNA was extracted with phenol-chloroform (V/V) and precipitated by the addition of ethanol (2 vol) and ~15μg of glycogen (1μl of Glycoblue 15mg/ml). RNA samples were treated with DNase I (Fermentas) for 45 min at 37°C. DNase I was then removed using phenol- chloroform extraction and RNA was again precipitated. The RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase (11 cycles). The TruSeq barcode sequences which are part of the 5' TruSeq sequencing adapter are included in Table 2. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. For Illumina NextSeq sequencing, the samples were pooled in approximately equimolar amounts. The cDNA pool in the size range of 200-550 bp was eluted from a preparative agarose gel. The cDNA pool was paired- end sequenced on an Illumina NextSeq 500 system using 2x50 bp read length. library strategy: MAPS