biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsAnne Stringernot applicableIllumina HiSeq 1000DNA-seqSalmonella enterica subsp. entericaSalmonella enterica subsp. entericahttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16844Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S CRISPRi library, growth with and without bile salts.biostudies-arrayexpressSequencing - Sequencing was performed on an Illumina Next-Seq Instrument (Wadsworth Center Applied Genomics Technologies Core).Sample Treatment - Cells are resuspended in LB + 100mM MOPS + 100ug/mL ampicillin, with or without 3% bile salt treatment. (sodium choleate, SIGMA S9875)Sample Treatment - no treatmentSample Collection - Sample collection and nucleic acid purification happen in the same step. After setting up initial culture, miniprep remaining cells from thawed aliquot (= \"start\"). Use Qiagen Spin Miniprep Kit -include Buffer PB wash, elute in 30 uL water. After 4.5 hrs growth and dilute to OD600=0.01, take 1.2 mL of culture and miniprep (same miniprep procedure) (= \"set 1\"). After 4.5 hrs growth, take 1.2 mL of culture and miniprep (same miniprep protocol) (= \"set 2\"). Qubit quantify all minipreps. Start and set 2 sample used for this experiment.Growth Protocol - Thaw glycerol stocks pAMD240 and pAMD241. Mix gently. Add 250 uL each glycerol stock to 12.5 mL LB+amp. Measure OD600. Dilute again 1 mL culture in 12.5 mL LB+amp. Record OD600. Grow o/n at 37deg/225 rpm. Next day set up 200 uL aliquots of cells + 50 uL 50% glycerol and flash freeze on dry ice. Thaw aliquot of frozen cells from \"14028s CRISPRi culture setup\" protocol. Dilute cells to OD600=0.01 (4 mL volume). Grow 4.5 hours at 37deg/225 rpm. Dilute to OD600=0.01 (4 mL volume). Grow 4.5 hours at 37deg/225 rpm.Library Construction - use 20 ng of miniprepped DNA (from \"miniprep\" sample extraction protocol) as template for PCR. Set up 4 x 50 uL PCR for each sample using Taq. Final concentration ultramers = 0.1 uM. Cycling: initial denaturation 95deg - 5 min; 10 cycles of 95deg-30 sec, 53deg-40sec, 68deg-1min; final extension 68deg-5 min; 4deg hold. Combine like PCRs and run 15 uL on acrylamide gel, 110V, 75min. Divide remaining PCR sample into 2 tubes and AMPure 0.8x clean. Elute each in 30 uL water, combine like samples, AMPure 0.8x clean again. Elute 11 uL. Qubit quantify.Growth Protocol - Thaw glycerol stock pAMD241 (14028s dCas3::thyA + Salmonella CRISPRi library). Mix gently. Add 250 uL each glycerol stock to 12.5 mL LB+ 100ug/mL ampicillin. Measure OD600. Dilute again 1 mL culture in 12.5 mL LB+/mL ampicillin, Record OD600. Grow o/n at 37deg/225 rpm. Next day set up 200 uL aliquots of cells + 50 uL 50% glycerol and flash freeze on dry ice. For experiment, thaw 3 aliquots frozen cells. Wash 2x in water, resuspend in 190 ul LB + 100mM MOPS + 100ug/mL ampicillin. Add 18.7 ul cells to 4 mL LB + 100mM MOPS + 100ug/mL ampicillin and add to 4mL LB + 100mM MOPS + 100ug/mL ampicillin + 3% bile salts. Grow 4.5 hours at 37C/225 rpm. Miniprep cells for “set1”. Dilute to OD600=0.01 (4 mL volume). Grow 4.5 hours at 37C/225 rpm. Miniprep cells for “set2”. Miniprep remaining cells form frozen aliquot for “start”Nucleic Acid Extraction - After setting up initial culture, miniprep remaining cells from thawed aliquot (= \"start\"). Use Qiagen Spin Miniprep Kit -include Buffer PB wash, elute in 30 uL water. After 4.5 hrs growth and dilute to OD600=0.01, take 1.2 mL of culture and miniprep (same miniprep procedure) (= \"set 1\"). After 4.5 hrs growth, take 1.2 mL of culture and miniprep (same miniprep protocol) (= \"set 2\"). Qubit quantify all minipreps. Start and set 2 sample used for this experiment.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesAnne Stringerfalse14028s CRISPRi Bile saltsSalmonella enterica subsp. enterica serovar Typhimurium str. 14028S CRISPRi library, growth with and without bile salts.2026-04-15T00:00:00Z2026-04-17T01:01:34.054Z2026-04-01T11:16:39.584ZE-MTAB-16844ERP191665EFO_0002944EFO_0004170EFO_0003789EFO_0002693EFO_0005518EFO_0004184EFO_0003969