title: GSM3594064: DK5955_B RibosomeProfiling; Bacillus subtilis; OTHER
library_construction_protocol: Following lysis, 25 A260 units of lysate were digested with 1500 units of S7 micrococcal nuclease (Roche 10107921001) for 1 hr at room temp after which the reaction was quenched by the addition of EGTA to a final concentration of 6 mM. The digested lysate was then applied to a 10%-50% sucrose gradient and centrifuged in a Ti-40 rotor at 35,000 rpm for 2.5 hrs at 4°C. 700 µL of fractions containing 70S ribosomes were denatured in 1% SDS and extracted once with an equal volume of hot acid phenol, once with an equal volume of room temp acid phenol, and RNA was precipitated with isopropanol. The precipitant was resuspended in 12 µL H2O and 25 µg RNA was mixed with 2X loading dye (10 mM EDTA, 30 μg/mL bromophenol blue, and 98% formamide) and resolved on a 15% polyacrylamide TBE Urea gen run at 250 V for 40 min. After staining the gel in SYBR Gold (Sigma S11494) for 3 min, products between ~15 and 40 bp were excised and subsequently gel extracted. RNA 3' ends were dephosphorylated with T4 poly-nucleotide kinase (Lucigen). Dephosporylated RNA was subsequently ligated to Linker 1 (IDT /5rApp/CTGTAGGCACCATCAAT/3ddC/)  with T4 RNA ligase 2, truncated (NEB) and products between 30-100 bp were gel extracted. Isolated RNA was then reverse transcribed using Superscript III (Invitrogen) with the reverse transcription primer (IDT 5′-(Phos)-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC-(SpC18)-CACTCA-(SpC18)-TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGC CTACAG-3). RNA was subsequently hydrolyzed by the addition of 2.2 µL 1N NaOH and incubation at 98C for 20 min. cDNA products were isolated by gel extraction and circularized using CircLigase (Epicentre). Circularized products were  used as a template for PCR reactions using Phusion Polymerase (NEB M0530S) with forward library primer (IDT 5′-AATGATACGGCGACCACCGAGATCTACAC-3′) and Indexed reverse library primer (IDT 5′-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGACTGGAGTTCAGACGTGTGCTCTTCCG-3′) where NNNNNNNN represents the barcode sequence unique to each library. After 6-10 cycles, two PCR reactions per sample with no apparent duplexed products after resolution of 2 μL on an 8% polyacrylamide TBE urea gel were pooled and DNA was purified with a QIAquick kit (Qiagen) and eluted in 20 µL H2O.
attributes.tag: GEO Accession
attributes.value: GSM3594064