SL RNA-seq of LdoS-infected macrophages after 24 hr (rep 2)
Poly A RNA was purified using the Dynabead mRNA purification kit                 (ThermoFischer, 61006.) 100ng mRNA was annealed to the first strand primer by adding                 2ul 20uM Random New Primer, 1ul 10mM RNA grade dNTPs, incubated at 65C for 5min then                 cooled to 4C. 1ul Superscript III, 4ul Superscript III First Strand Buffer, 1ul 0.1M                 DTT was added to each sample and samples incubated at 25C for 5min, 50C for 60min,                 and 70C for 15min. Samples were treated with RNAse H by adding 2units RNAse H, 2.5ul                 NEB RNAse H Buffer, and 2.1ul Nuclease Free Water. Samples incubated at 37C for 20                 minutes. A 1.8X Ampure XP purification was performed by adding 45ul Ampure XP beads                 to each sample, incubated at room temperature for 10 minutes, and placed on magnet                 until supernatant was clear. Beads were washed twice with freshly prepared 80% EtOH                 and dried. Sample was eluted from beads in 25ul 10mM Tris, pH 8. Second strand                 synthesis was performed by adding 5ul 10X NEBbuffer 2, 3ul 10uM SL primer, and 20ul                 Nuclease free water. Samples were heated to 95C and allowed to cool to RT for 30min                 to bind SL primer. To create the second strand product, 1ul NEB Klenow Fragment and                 1ul 10mM dNTPs was added to each sample and they were incubated at 37C for 60min.                 The Qiagen Qiaquick PCR purification kit was used to purify the second strand                 product. Product was amplified using 2x KAPA Hifi DNA polymerase Mastermix, 1ul 10uM                 P5 primer, 1ul 10uM P7 primer, and water to 50ul. PCR product was purified using the                 Qiagen PCR purification kit and sequenced on the NextSeq 500 using the SL Sequencing                 Primer (CACCGAGATCTCACTCTTTCCCTA CATCAGTTTCTGTACTTTATTG).