GSM3724947: LogPhase Fraction 19; Homo sapiens; OTHER
Cells were washed and detached with 10 ml ice cold PBS supplemented with 100 µg/ml cycloheximide / 15cm culture dish and collected at 1000 rpm for 5 minutes at 4°C. Cells were lysed in 250 µl lysis buffer (100mM KCl, 20mM Tris pH 7.5, 5mM MgCl2, 1mM DTT, 1mM Leupeptin, 1mM Pepstatin, 1mM Aprotinin, 0,1 mM PMSF, 0,1 mM AEBSF, 0,5% NP40, 100 µg/ml cycloheximide, 40 U/ml RNAsin (Promega)) for 10 minutes on ice. Cell debris were pelleted at 10k rpm for 10 minutes in an Eppendorf tabletop centrifuge. Library preparation was performed according to the Illumina TruSeq stranded mRNA Sample Preparation Guide with 300 - 500 ng of input RNA and 15 PCR cycles. All libraries were dual-indexed during PCR. Libraries were pooled equimolar and sequenced on an Illumina NextSeq 500 with a read length of 75 nt.
GEO
GSM3724947