GSM8149777: Ctrl_01_C21; Danio rerio; RNA-Seq
The cells are sorted with an Aria III FACS into a 384 well plate pre-loaded with 0.5 µl freshly prepared lysis buffer (0.2 % Tween-20 solution with 4 U/µl murine RNase inhibitor (NEB)). After the sort, the plate is shortly centrifuged and lysed cells stored at -80°C.After thawing the plate, the cells are immediately mixed with 0.5 µl of a primer mix containing 5 mM dNTP (Invitrogen), 0.5 µM dT-primer*, 2 U RNase Inhibitor (NEB), ERCC spike in mix with a final dilution of 1:2*107. RNA is denatured for 3 minutes at 72 °C, followed by reverse transcription at 42 °C for 90 min after filling up to 1.5 µl with RT buffer mix for a final concentration of 1x superscript II buffer (Invitrogen), 1 M betaine, 5 mM DTT, 6 mM MgCl2, 1 µM TSO-primer*, 2.3 U RNase Inhibitor and 25 U Superscript II. After synthesis, the reverse transcriptase is inactivated at 70°C for 15 min. The cDNA is amplified using Kapa HiFi HotStart Readymix (Peqlab) at a final 1x concentration and 0.1 uM UPprimer under following cycling conditions: initial denaturation at 98°C for 3 min, 23 cycles [98°C 20 sec, 67°C 15 sec, 72°C 6 min] and final elongation at 72°C for 5 min. The amplified cDNA is purified using 0.6x volume of hydrophobic Sera-Mag SpeedBeads (GE Healthcare) resuspended in a buffer consisting of 10 mM Tris, 20 mM EDTA, 18.5 % (w/v) PEG 8000 and 2 M sodium chloride solution and DNA is eluted in 12 ul nuclease free water. The concentration of the samples is measured with a Tecan plate reader Infinite 200 pro in 384 well black flat bottom low volume plates (Corning) using AccuBlue Broad range chemistry (Biotium) For library preparation up to 700 pg cDNA (maximal 2 µl) is desiccated and rehydrated in 1 ul Tagmentation mix (1x TTBL buffer, 0.1 ul Tagment DNA Enzyme; from TruePrep DNA Library Prep Kit V2 for Illumina; Vazyme) and tagmented at 55°C for 5 min. Subsequently, Illumina indices are added during PCR (72°C 3 min, 98°C 30 sec, 13 cycles [98°C 10 sec, 63°C 20 sec, 72°C 1 min], 72°C 5 min) with 1x concentrated KAPA Hifi HotStart Ready Mix and 0.17 µM dual indexing primers. After PCR, libraries are quantified with AccuBlue Broad range chemistry, equimolarly pooled and size selected with 0.6x(right)/0.9x (left) volume of rebuffered Sera-Mag SpeedBeads. This is followed by Illumina sequencing on a Novaseq6000 (50 bp PE, S1 flowcell) aiming at an average sequencing depth of 0.6 mio reads per cell. dT-primer: C6-aminolinkerAAGCAGTGGTATCAACGCAGAGTCGAC TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN, where N represents a random base and V any base beside thymidine; TSO-primer: AAGCAGTGGTATCAACGCAGAGTACATrGrGrG, where rG stands for ribo-guanosine; UP-primer: C6-aminolinker- AAGCAGTGGTATCAACGCAGAGT