GSM4932435: KO04; Homo sapiens; RNA-Seq
Single cells were index sorted using a BD FACS Influx into 384-well hardshell plates (BioRad) that were pre-filled with 5 µL of light mineral oil (Sigma Aldrich) and 50 nL of lysis buffer [22 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 5.5 mM CaCl2, 165 mM NaCl, 1.1 % Triton X-100, 2.2 U/µL RNaseIN Plus (Promega), 0.11 mg/mL Cyclohexamide (Sigma Aldrich)]. After sorting, plates were spun down at 2000 ×g for 2 minutes and kept on wet ice until all plates were ready for further processing. No RNA purification was performed. To generate ribosome-protected footprints, 50 nL of Micrococcal Nuclease (MNase, 10500 U/µL, New England Biolabs) was added to each well, and plates were incubated at 37 °C for 30 minutes. In order to stop digestion, 50 nL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and plates were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C. After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 50 nL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 264 nL of 3' ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3' adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3' ligation products by adding 50 nL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 156 nL of 5' ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5' adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 771 nL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, single-cell libraries were indexed during PCR by first transferring 150 nL of 20 µM unique forward index primers (Integrated DNA Technologies) and 3.2 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to each well. Plates were then incubated at 98 °C for 30 s followed by 10 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until pooling. After library construction the plates were pooled and purified. The contents of each plate were first collected in VBLOK200 reservoirs (Click Bio) by centrifuging at 2000 ×g for 2 min. The aqueous phase (~1.9 mL per plate) was separated from the light mineral oil by centrifugation, and concentrated to approximately 500 µL using n-butanol (Sigma Aldrich) and diethyl ether (Sigma Aldrich). Product was then cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 5× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 50 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 10 cm 7 % polyacrylamide gel at 200 V for ~6 h, and the ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in elution buffer [5:1 LoTET:7.5 M ammonium acetate (Sigma Aldrich)]. Finally, eluate was precipitated in ethanol. adapters: barcode: CAGATC; 5' Adapter: rCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUr(N1:25252525)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1); 3' Adapter: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/