title: GSM3132064: Cell6 (small RNA-seq); Homo sapiens; ncRNA-Seq
library_construction_protocol: Cells were washed once with cold PBS (Life Technologies) and diluted to < 1000 cells/ml. Prior to picking single cells, 10 µl of lysis buffer (0.25% Triton-X100, 4 units (U) recombinant RNase Inhibitor, Takara) was deposited in each PCR tube. To ensure single cell resolution, we performed most experiments by hand-picking of single cells under microscope. The diluted cell mixture was placed on the cover of a petridish, and single cells without close neighbours were manually picked by using a 10 µl pipette, with each cell contained within less than 1 µl of volume. Single cells were then added into the lysis buffer. The single-cell lysates were stored at −80 °C. After thawing, the lysates were incubated at 72 °C for 20 min to release small RNAs for reaction. Small RNA library preparation: Single-cell or half-cell lysate (5 µl) was subjected to 3' adaptor ligation by adding 5.24 µl of 3' adapter ligation reaction mixture (1 pmole 3' adapter, 1.66 µl 50% PEG 8000, NEB, 250 U T4 RNA Ligase 2 truncated KQ, NEB M0373S, 0.83 µl 10× T4 RNA ligase buffer, NEB, 20 U recombinant RNase Inhibitor, Takara) and the reaction was incubated at 30 °C for 6 hours followed by 4 °C for 10 hours. Next, 5 µl adaptor-digestion mixture was added (10 pmole RT primer, 5 U Lambda exonuclease, NEB, 5 U 5' deadenylase, NEB) and the reaction was incubated at 30 °C for 15 min followed by 37 °C for 15 min. Next, 5 µl 5' adapter ligation reaction mixture was added (10 pmole 5' adapter oligo, 10 U T4 RNA ligase 1, Thermo Fisher, 1 µl T4 RNA ligase buffer, Thermo Fisher, 2 µl 50% PEG 8000, NEB) and incubated at 37 °C for 1 hour. Reverse transcription reaction was performed with three steps and was performed with M-MLV reverse transcriptase (RT) (Invitrogen 28025-013). First, 5 µl of RT reaction mix was added (10 pmole RT primer) and the reaction was incubated at 65 °C for 5 minutes. Second, the mix of 2.1 µl 5x First Strand buffer (Invitrogen), 0.3 µl H2O and 0.8 µl of 0.1M DTT (Invitrogen) was added and incubated at 42 °C for 30 minutes. Third, 3.3 µl of RT reaction was added (0.6ul M-MLV reverse transcriptase, 1 µl 5x First Strand buffer (Invitrogen), 0.3 µl H2O, 0.8 µl of 0.1M DTT, Invitrogen and 0.6 µl 10mM dNTP mix, Invitrogen) and the reaction was incubated at 42 °C for 2 h. The first PCR amplification was carried out by adding 35 µl of the reagents (3.5 µl 10xThermoPol Reaction Buffer (NEB), 0.7 µl 10mM dNTP mix (Invitrogen), 35 pmole RT primer oligo, 35 pmole PCR primer oligo, 0.5 µl Taq DNA Polymerase (NEB), 23.3 µl H2O) and incubating at 98 °C for 30 seconds followed by 13 cycles of 98 °C for 10 seconds, 60 °C  for 30 seconds and 72 °C  for 30 seconds and a final incubation at 72 °C for 5 minutes. At last, 1 µl of the amplified product was transferred to a fresh tube and to a 25 µl of second PCR reaction consisting of 10 µM indexed primer, 10 µM RP1 primer, 2.5 µl 10x ThermoPol Reaction buffer, 0.5 µl Taq DNA Polymerase (NEB), 0.5 µl 10mM dNTP mix (Invitrogen), followed by a 30 second incubation at 98 °C, 13 cycles of 98 °C for 10 seconds, 67 °C for 30 seconds and 72 °C for 30 seconds and a final incubation at 72 °C for 5 minutes. PCR product of ~140 bp were gel-purified after 2nd PCR on a 8% acrylamide gel. Gels were prepared by adding 9.3 ml Protein Gel Mix (American Bioanalytical AB13021), 3.6ml 10x TBE, 23.1 ml water, 600 µl 10% ammonium persulfate (Sigma Aldrich), and 30 µl TEMED (American Bioanalytical). Samples were prepared by adding 1/5 volume of 6X Gel loading dye purple (New England BioLabs). Electrophoresis was performed at 140V for 80 minutes. After electrophoresis, the gel was stained in 15 ml water containing 1.5 µl GelStar Nucleic Acid Gel Stain (LONZA) for 15 minutes. All barcoded small RNA libraries were sequenced on an Illumina HiSeq 2000 instrument.