Time Dependent transcriptome study of Bacillus Sps Biofilm Cells.
Library preparation was performed at Genotypic Technology’s Genomics facility. 5µg of QC passed total RNA was taken for ribo depletion, performed using RiboMinus Bacterial Kit (Invitrogen Catalog. no. K1550-04). 100ng of qubit quantified ribodepleted RNA was taken for lib prep. Transcriptome library for sequencing was constructed according to the Illumina TruSeq RNA library protocol outlined in “TruSeq RNA Sample Preparation Guide” (Part # 15026495 Rev. F). Briefly, the Ribodepleted RNA was fragmented for 8 minutes at elevated temperature (94oC) in the presence of divalent cations and reverse transcribed with Superscript III Reverse transcriptase by priming with Random Hexamers. Second strand cDNA was synthesized in the presence of DNA Polymerase I and RnaseH. The cDNA was cleaned up using HighPrep PCR (MAGBIO, Cat# AC-60050). Illumina Adapters were ligated to the cDNA molecules after end repair and addition of A base. SPRI cleanup was performed after ligation. The library was amplified using 8 cycles of PCR for enrichment of adapter ligated fragments. The prepared library was quantified using Qubit and validated for quality by running an aliquot on High Sensitivity D1000 Tapestation (Agilent).