title: GSM3100775: H9 ESCs_Aba-seq; Homo sapiens; OTHER
library_construction_protocol: ~50,000 cells were suspended in 10 µL of lysis buffer (100 µg Qiagen Protease, 0.04% Triton X-100, 1x NEB CutSmart buffer) and incubated at 50 °C for 15 h, 75 °C for 20 min and 80 °C for 5 min. Then 10 µL of 1x NEB CutSmart buffer, 10 U NEB T4-BGT, 2.5x NEB UDP-Glucose was added to glucosylate 5-hmC sites in the genome, and the sample was incubated at 37 °C for 16 h.  Next, 10 µL of protease mix (100 µg Qiagen Protease, 1x NEB CutSmart) was added and incubated at 50 °C for 5 h, 75 °C for 20 min and 80 °C for 5 min.  10 µL of digestion mix (10 U AbaSI, 1x NEB CutSmart) was added and incubated at 25°C for 2 h, and 65 °C for 20 min.  Next, 1 µL of 0.5 µM ds-adaptor was added, followed by 9 µL of ligation mix (2,000 U T4 DNA Ligase, 1x T4-ligase buffer, 3.33 mM ATP) and each sample was incubated at 16 °C for 16 h.  The ds-adaptor sequences are described in scAba-seq (Mooijman et al., 2016).  DNA clean-up was then performed with 0.825x Agencourt Ampure XP beads and eluted in 25 µL of nuclease-free water.  The samples were vacuum centrifuged to a volume 8 µL.  12 uL of in vitro transcription mix was added (2 µL of each ribonucleotide, 2 µL of T7 buffer, 2 µL T7 enzyme mix) and incubated at 37 °C for 13 h.  Library preparation starting from amplified RNA was performed as described in the CEL-Seq2 protocol with the following minor modifications (Hashimshony et al., 2016).  RNA was fragmented by adding 5 µL of fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500mM KOAc, 150 mM MgOAc) at 94 °C for 2 min and immediately placed on ice. 2.5 µL of fragmentation stop buffer (0.5 M EDTA) was added to quench the reaction.  Next, RNA clean-up was performed using 0.825x Agencourt RNAClean beads and finally resuspended in 22 µL of nuclease-free water.  Thereafter, all library preparation steps were performed as described in the CEL-Seq2 protocol (Hashimshony et al., 2016).
attributes.tag: GEO Accession
attributes.value: GSM3100775