GSM6914701: 3-prime-rnaseq-TS-stationary3; Borreliella burgdorferi; RNA-Seq
RNA was isolated using TRIzol. RNA was prepared for sequencing as described previously (Adams et al., 2021). Any contaminating DNA in the samples was removed by treating 15 µg of RNA with 10 U of DNase I (Roche) for 15 min at 37˚C in the presence of 80 U of recombinant RNase inhibitor (Takara Bio). Next, RNA was purified by mixing the sample with an equal volume of phenol stabilized:chloroform:isoamyl alcohol (25:24:1) and centrifugation at maximum speed in Heavy Phase Lock Gel tubes (5 PRIME). A volume of chloroform, equal to the original sample volume, was added to the same Heavy Phase Lock Gel tubes and spun again. The aqueous layer was removed and ethanol precipitated. RNA pellets were reconstituted in 10 µl DEPC H2O and analyzed using an Agilent 4200 TapeStation System to ensure DNase-treated RNA was at high quality. Term-seq libraries were prepared using a modified version of the RNAtag-seq methodology (Shishkin et al., 2015), based on the previously published Term-seq methodology (Dar et al., 2016). 1.5 µg of DNA-free RNA was first ligated at the 3′ end with 150 µM barcoded oligonucleotide adapters which were 5′phosphorylated and dideoxycytidine 3′ terminated. RNA and 3′ adapters were incubated at 22˚C for 2.5 hr with 51 U of T4 RNA Ligase I (NEB) and 12 U of recombinant RNase inhibitor (Takara Bio) in 1X T4 RNA Ligase Buffer (NEB), 9% DMSO, 20% PEG 8000, and 1 mM ATP. 3′ ligated RNA was cleaned by incubating with 2.5X volume of RNAClean XP beads (Beckman Coulter) and 1.5X volume of isopropanol for 15 min, before separation on a magnetic rack. Bead-bound RNA was washed with 80% ethanol, air dried, and resuspended in DEPC H2O. RNA-containing-supernatants were removed and the same RNAClean XP bead cleanup protocol was repeated, with a final DEPC H2O elution of 9.5 µl. RNA was fragmented by incubating 9 µl of cleaned-up RNA with 1X Fragmentation Reagent (Invitrogen) for 2 min at 72˚C, followed by an addition of 1X Stop Solution (Invitrogen). Samples were stored on ice following individual fragmentation of each sample. Fragmented-RNA was pooled together and cleaned using the RNA Clean and Concentrator-5 kit (Zymo) according to the manufacturer's instructions. Library construction continued following the bacterial-sRNA adapted, RNAtag-seq methodology starting at the rRNA removal step (Melamed et al., 2018). Term-seq RNA libraries were analyzed on a Qubit 3 Fluorometer (Thermo Fisher Scientific) and an Agilent 4200 TapeStation System prior to paired-end sequencing using the HiSeq 2500 system (Illumina).