title: GSM9268328: B. fragilis microSPLiT library F3 12k; Bacteroides fragilis; RNA-Seq
library_construction_protocol: Fixed cells were spun down at 10,000 rcf for 10 min at 4°C, resuspended in cold 100mM Tris-HCl+RI (100mM Tris-HCl pH=8, 20U/mL SUPERase-In RNase Inhibitor (Thermo Fisher Scientific)), and spun down again in the same conditions. Cells were resuspended in 250µL permeabilization solution (0.04% Tween-20 in PBS), incubated for 3 min on ice, and after 1mL addition of PBS+RI (1x PBS, 6U/mL SUPERase-In RNase Inhibitor, 24U/mL Enzymatics RNase Inhibitor (Qiagen)), spun down at 10,000 rcf for 10 min at 4°C. Cells were aspirated and resuspended in 200µL digestion mix (100mM Tris-HCl pH=7, 50mM EDTA, 7.5µg of lysozyme (Thermo Fisher Scientific), 250U/mL SUPERase-In RNase Inhibitor), incubated for 15 min at 37°C, immediately added to 1mL PBST (0.01% Tween-20 in PBS+RI), and centrifuged at 10,000 rcf for 10 min at 4°C. Samples were then resuspended in 50µL PBS+RI per sample post-permeabilization. Cells were counted by hemocytometer, and 7 million cells were taken per plate for polyadenylation. 50µL polyadenylate polymerase I solution (1x E. coli Poly(A) polymerase buffer, 800U/mL SUPERase-In RNase Inhibitor, 50mM ATP, 25U of E. coli Poly(A) polymerase) was added, and each sample was incubated at 37°C for 30 min, spun down with 1mL PBST, and resuspended in PBS+RI. Cells were counted again for verification of the number of cells per plate. Reverse transcription (RT) mix was created on ice with final concentrations of 1X reverse transcription buffer, 0.25U/µL Enzymatics RNase Inhibitor, 0.25U/µL SUPERase-In RNase Inhibitor, 500µM dNTPs, 10U/µL Maxima H Minus Reverse Transcriptase, and 7.5% PEG8000 with a final volume of 1320µL for a 96 well plate. Reverse transcription mix was added (12µL) to wells, already containing 4µL mixture of 12.5µM random hexamer and 12.5µM 15dT primer in each well; cells were vortexed and 4µL suspension was added to wells for a final volume of 20µL. Each sample was loaded into different wells of the reverse transcription plate to demultiplex them during analysis. Plates were incubated at 23°C for 10 min and at 50°C for 50 min at 500 rpm. Then, 40µL PBST was added to each well on ice, samples were pooled in a single tube with 24.96µL 10% Tween-20, and the total reaction was centrifuged for 10 min at 10,000 rcf at 4°C. The sample was aspirated and resuspended in 2.2mL PBS+RI and disaggregated with both a 10µm and 1µm pluriStrainer filters. The composition of the 96-well plate and barcoded oligonucleotides used for RT were as previously described in in Gaisser and Skloss et al., 2024. The ligation master mix was made on ice, containing final concentrations of 1X T4 Ligase Buffer 10X, 0.58U/µL Enzymatics RNase Inhibitor, 0.05U/µL SUPERase-In RNase Inhibitor, 7.5% PEG8000, and 8U/µL T4 DNA Ligase, with a final volume of 2040µL. 2mL cell suspension was added to the ligation mix and 40µL aliquots were transferred into each well of a Round 2 ligation barcode plate before incubating for 30 min at 37°C and adding 10µL of round 2 blocking solution (26.4µM ​BC_0340​, 2.5X Ligase Buffer). The plate was incubated again for 30 min at 37oC, and cells were pooled, disaggregated, and barcoded again (Round 3 blocking solution: 11.5µM​ BC_0066 , 125mM EDTA). After the final filtering step, cells were washed (4000µL 1X PBS, 40µL 10% Tween-20, 10µM SUPERase-In RNase Inhibitor) and aliquoted into libraries with PBS+RI to a final volume of 50µL. Again, the compositions of the 96-well plates and oligonucleotides used for barcoding step were as previously described in in Gaisser and Skloss et al., 2024. Aliquoted libraries were mixed with 50µL lysis buffer (20mM Tris-HCl pH=8, 400mM NaCl, 100mM EDTA pH=8, 4.4% SDS) and 10µL of 20mg/mL Proteinase K for protein digestion prior to incubation at 55°C for 2 h with 500 rpm shaking. The resulting biotinylated cDNA was captured on MyOne C1 Dynabeads. For template switching, a common 5' sequence was added to full-length cDNA by resuspending the beads in 200µL Template Switch Mix (1X Maxima RT Buffer, 7.5% PEG8000, 1mM of each dNTP, 500U/mL SUPERase-In RNase Inhibitor, 2.5µM TSO primer, 10U/µL Maxima H minus RT) and incubating at room temperature for 20 min at 600 rpm before further incubation at 42°C for 90 min with agitation. Then, beads were washed in 250µL Tris-T+RI (10mM Tris-HCl, 0.1% Tween-20, 50U/mL SUPERase-In RNase Inhibitor), decanted, and resuspended again with 100µL per sample of terminal deoxynucleotidyl transferase tailing (TdT) master mix (1X TdT buffer, 0.25mM CoCl2, 1.2mM dCTP, 0.06mM ddCTP, 2U/µL TdT enzyme (NEB), 0.1U/µL RNase H (NEB)). Following incubation for 1 h at 37°C and a wash with Tris-T+RI, cDNA was amplified via ​qPCR​ using primers BC_0108/M_0002 and BC_0062, fragmented, and ligated with adapters prior to Illumina Sequencing as was described previously in Gaisser and Skloss et al., 2024.  Sequencing was performed on NovaSeq X Plus Series (PE150, Novogene).