GSM5888798: Grad-seq E. faecalis fraction 10; Enterococcus faecalis V583; OTHER
Gradient samples were denatured with 1% SDS and RNA was PCI extracted, and ethanol precipitated. 5 µl of the resulting samples were diluted in 45 µl DEPC-treated water. 10 µl were mixedwith 10 µl of a 1:100 diluted ERCC spike-in mix 2 (Thermo Fisher) and subjected to library preparation. In RIP-seq, RNA-protein interaction was denatured by 1% SDS and PCI extraction. RNA was precipitated with ethanol and submitted to library preparation. The RNA samples were fragmented using ultrasound (4 pulses of 30 s at 4°C) followed by 3′-adapter ligation. First strand cDNA synthesis was performed using M-MLV reverse transcriptase. After purification, the 5′-Illumina TruSeq sequencing adapters were ligated to the 3′end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10–20 ng/µl by high-fidelity DNA polymerase and purified with Agencourt AMPure XP kit (Beckman Coulter). cDNA samples were pooled with according to input concentration, a size range of 200–550 bp was eluted from a preparative agarose gel. This size-selected cDNA pool was sequenced on an Illumina NextSeq 500 system using 75 ntsingle-end read length.