GSM9035189: L. lactis subps. cremoris MG1363_control_rep1; Lactococcus cremoris subsp. cremoris MG1363; RNA-Seq
Bacteria were mixed with 1/8 volume of ice-cold STOP solution (95% ethanol and 5% acid phenol) and centrifuged at 1,800 × g for 20 min at 4°C. Cells were ground to a fine powder using liquid nitrogen and total RNA was extracted with RNAzolRT (RN 190) (Molecular Research Center, Inc.). 50–500 nt RNA was cut out of 8% denaturing PAA gel. 50–500 nt RNA was treated with DNase I, RNase free (Thermo Scientific) and recovered from the reaction using RNA Clean & Concentrator-5 Kit (Zymo Research). 5S rRNA was depleted using the modified RiboMinus Transcriptome Isolation Kit, bacteria (Invitrogen) where 4 μl of 100 μM mix of three probes specific to L. lactis 5S rRNA were used. After ethanol precipitation RNA was treated with RNA 5′ Pyrophosphohydrolase (RppH) (New England Biolabs), recovered with RNA Clean & Concentrator-5 Kit and 165 ng were taken for further processing using NEXTflex Small RNA-Seq Kit v3 (PerkinElmer) with following modifications: at Step E, incubation at 42°C was extended to 60 min; at Step G, 16 PCR cycles were performed; and Steps F and H1 were carried out as described in alternative protocol for Preparing Libraries without Size Selection.