title: GSM5471122: E.  coli, K12 strain, clone 2, bisulfited, ABBS primer; Escherichia coli; Bisulfite-Seq
library_construction_protocol: Genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen, Germany) following the manufacturer's protocol. 3μg of E. coli or 10μg of K562 genomic DNA spiked with 1% of unmethylated Lambda DNA was bisulfite-converted with Zymo Lightning kit (Zymo Research, CA) following the manufacturer's  instructions, then eluted in 2x10μl of Tris-HCl 10mM pH8.0 (of note, desulfonation was performed for 20mins, on column). An additional 15-minute sonication was achieved using PIXUL Multisample sonicator (Active Motif, CA) with the default settings. DNA was heated at 95C for 3 minutes then placed on ice and quantification was done using Nanodrop (Thermo Fisher Scientific, MA) while profiles were measured with RNA TapeStation (Agilent, CA) to ensure for proper sonication. Bacterial non-converted DNA was sonicated for 30 minutes using PIXUL Multisample sonicator with the default settings and DNA was further heated at 95C for 3 minutes and stored at -80C.  Second strand synthesis was performed as follow: 100ng of genomic DNA was diluted in Neb 1x NEBuffer 2, supplemented with 0.25mM dNTPs and 6.5μM (K562 cells) or 0.65μM (E. coli K12 cells) ABBS primers (NNNNNSuperG) or WGBS primers (NNNNNN) in 29 μl, then heated at 94C for 2 minutes and rapidly placed on ice for 3-5 minutes. One microliter of Neb Klenow exo minus (5 units/μl) was added to the reaction that was gradually heated from 4C to 37C at a rate of 0.1C/second. Samples were then incubated for 30 minutes at 37C and purified with 45μl of Ampure XP beads (Beckman Coulter, CA). DNA was eluted in 20μl Tris-HCl 10mM pH8.0 supplemented with 0.05% Tween-20 and quantified with Qubit 2.0 dsDNA HS kit (Thermo Fisher Scientific, MA).  Double stranded library preparation was performed either with Neb NEBNext® UltraTM II DNA Library Prep Kit for Illumina or Active Motif Next Gen DNA Library Kit, following manufacturers' recommendations, with the exception that libraries amplification was performed using KAPA HiFi HotStart Real-Time Library Amp Kit (Roche, Switzerland). After a final purification using 0.75 volumes of Ampure XP beads, libraries were resuspended in 12μl of Tris-HCl 10mM pH8.0 supplemented with 0.05% Tween-20 and quantified with Qubit 2.0 dsDNA HS kit while profiles were measured with HS dsDNA TapeStation to calculate molarity