HiSeq X Five paired end sequencing; RNA-Seq of mouse embryonic stem cells after knockout and re-introduction of Sall4a
RNA-Seq of mouse embryonic stem cells after knockout and re-introduction of Sall4a
To generate cell lines expressing a transgene of interest (Sall4 or EGFP cDNA) under a doxycycline-inducible promoter, Sall4 knockout ESCs were modified using the PiggyBac (PB) transposon system. 1x10^6 Sall4 knockout ESCs were transfected with two PiggyBac vectors (“PB-(TetO)8-Sall4-PGK-HygromycinR” or “PB-(TetO)8-EGFP-PGK-HygromycinR” + “PB-Tet-On 3G-IRES-ZeocinR”), together with a third plasmid expressing hyperactive PB transposase 70 (pCMV-hyPBase). 48h post-transfection, ESCs were selected for 12 days with 200µg/ml hygromycin (doxycycline-inducible Sall4/EGFP construct) and 100µg/ml zeocin (Tet-On 3G transactivator construct). This experiment was repeated three times to obtain independent replicates for each cell line (Sall4 or EGFP). During selection, no doxycycline was added to the medium in order to prevent Sall4/EGFP expression. To induce Sall4/EGFP expression, cells were treated for 48h with 1µg/ml doxycycline (freshly prepared).  For RNA-seq in ESCs, all cell lines were seeded at the same density in 6-well plates, in three or four independent replicate experiments for each sample. Following two days of culture, total RNA was extracted using the AllPrep DNA/RNA kit (Qiagen) or the RNeasy Plus Mini kit (Qiagen), following manufacturer's instructions. Eventual contaminating genomic DNA was removed by DNase treatment. Before library preparation, equal amounts of either RNA sequins (Garvan Institute of Medical Research, Australia) or ERCC (Invitrogen) spike-in mix were added to each sample. Ribosomal RNA-depleted RNA-seq libraries were prepared using either the ScriptSeq Complete Gold Kit (Illumina) or the KAPA RNA Hyperprep Kit (Roche ref. 08098131702) together with indexed adapters, following manufacturer's instructions. RNA-seq libraries with unique barcodes were pooled in equimolar amounts and sequenced using the Illumina HiSeq 2500 (Wellcome Sanger Institute, UK), HiSeq X (Novogene Europe, UK) or NextSeq 500 (EMBL GeneCore facility, Germany) platforms.