title: GSM7669594: BC2; Mus musculus; RNA-Seq
library_name: GSM7669594
library_construction_protocol: Total RNA from oocytes was isolated using PicoPure RNA isolation kit (Thermo Fischer Scientific, Cat# KIT0204) according to manufacturer's protocol with following adjustments: groups of isolated oocytes were placed in 10 µl of provided Extraction Buffer in PCR tubes and incubated for 30 min in 42 °C on thermoblock. Samples were then stored in –80 °C. Before proceeding to next step, samples were pooled for total of 30 oocytes per sample and amount of provided ethanol used was adjusted depending on final sample volume. RNA was eluted with 11 µl of Elution Buffer provided. DNA was removed from final sample by DNase treatment using TURBO DNA-free Kit (Thermo Fisher Scientific, Cat# AM1907) according to manufacturer's protocol. DNA-free RNA was stored in –80 °C. cDNA was produced using SuperScript III reverse transcriptase (Thermo Fisher Scientific, Cat# 18080093) according to manufacturers protocol with oligo(dT) priming and addition of ERCC RNA Spike-InMix (final dilution: 1:100000; Life Technologies, Cat# 4456740) for first-strand synthesis from oocyte-isolated RNA and RiboLock RNase inhibitor (40 units; Thermo Fischer Scientific, Cat# EO0382) for second strand synthesis in all samples. Output material was purified in 1 volume of AMPure XP beads, eluted in 20 µl nuclease-free water and second strand synthesis reaction was set up by addition of following components: Second Strand Buffer (NEB, Cat# B6117S), RNase H (10 units; NEB, Cat# M0297S), E. coli DNA ligase (10 units; NEB, Cat# M0205S), E. coli DNA polymerase (50 units), dNTPs (final conc. 0.4 mM) and nuclease-free water for a final reaction volume of 50 µl. Reaction was incubated in 16oC overnight. Final cDNA output was purified in 1 volume AMPure XP beads and eluted with 10 µl of nuclease-free water. Tagmentation reaction was performed following published protocols63,64 with various steps and amount of enzyme used optimized for use of homemade batch of Tn5 transposase produced in our laboratory. Briefly: 100 µM Tn5ME-A and Tn5ME-B oligonucleotides in annealing buffer (50 mM NaCl, 40 mM Tris-HCl pH = 8) were mixed 1:1 with 100 mM Tn5MErev oligonucleotide and incubated in thermocycler in 95°C for 5 min and 65°C for 5 min, and finaly cooled to 4oC and stored at -20 oC (with slow cooling between each step). Tn5 (0.25 mg/ml) was loaded with linker oliguncleotides Tn5ME-A/Tn5MErev and Tn5ME-B/Tn5MErev (see Supplemental Table 1 for full sequences) by mixing of 10 µl Tn5 with 0.5 µl of both linkers (0.35 µM) and incubating for 45 min in 23oC with shaking at 350 rpm. Right before the reaction setup loaded Tn5 was diluted 10 times with nuclease-free water. 10 µl of freshly prepared tagmentation buffer (20mM Tris-HCl ph 7.5; 20  mM MgCl2; 50 % dimethylformamid)  were mixed with 5 µl of diluted Tn5 and 5 µl od cDNA, incubated for 3 min at 55oC in preheated thermocycler and cooled to 10oC for 1 min. Reaction was inactivated by addition of 5 µl 0,2% SDS and incubation for 5 min at RT. Tagmented cDNA was purified in 1.25 volume of AMPure XP beads and eluted in 10 µl of nuclease-free water. For library amplification KAPA HiFi HotStart ReadyMix 2x (Roche, Cat# KK2602) with addition of 5% DMSO was used. 5 µl of tagmented cDNA was used for 15 µl reaction volume. Reaction was run in thermocycler with following program: 72oC – 15 min., 95oC – 30 s., 15 cycles: (98oC – 20s., 58oC – 15s., 72o – 30s.), 72oC – 3 min., 4oC - hold. Number of cycles yielding best results was determined experimentally and was further individually adjusted for each batch of prepared cDNA.