title: GSM5368899: 4-cell mouse embryos - Library 1; Mus musculus; OTHER
library_construction_protocol: Embryos were gently flushed out of the infidubulum of E2.5 pregnant mice using warm M2 medium. Embryos were then manipulated in 4-ring IVF dishes coated with RNase-free BSA because, once dissociated, cells stick to plastic. Embryos were washed in PBS-0 and in Tyrode's acid to remove the zone pellucida, then placed in a 1/3 dilution of Tryple select GIBCO A12177-01 (stock solution is referred by Gibco as x10 concentrated) and placed on the warm plate for 2 minutes. We then used glass capillaries of different diameters to dissociate the embryo into 2-3 clusters. Cells were then progressively extracted from each cluster, one after the other, using glass capillaries. Every single cell that is released from the clusters is immediately placed into a well of a 384-well plate containing lysis buffer. 4 μL Vapor-Lock (Qiagen) is dispensed manually into each well of a 384 well plate using a multichannel pipet. 0.2 μL of lysis buffer (1x NEB buffer 4, 0.1 μg Qiagen protease) is dispensed at 12 psi pressure into each well using the NanoDrop II liquid-handling robot. Single cells are deposited  into each well and the plate is incubated at 50°C for 3 hours, 75°C for 20 min and 80°C for 5 min. 0.2 μL of Glucosylation mix (1x NEB buffer 4, 2 U NEB T4-BGT, 1x NEB UDP-Glucose) is added and the plate is incubated at 37°C for 18 hours. Next, 0.2 μL of lysis buffer is added and the plate is incubated at 50°C for 3 hours, 75°C for 20 min and 80°C for 5 min. 0.2 μL of AbaSI (1x NEB buffer 4, 2 U NEB AbaSI) is added and the plate is incubated at 25°C for 1 hour and 65°C for 20 min. 0.2 μL of 0.01 μM adapter is added, followed by 0.2 μL of ligation mix (80 U T4-Ligase, 2X T4-ligase buffer, 0.6 mM ATP) and the plate is incubated for 16 hours at 16°C. Contents of all the wells with different adapters are pooled and incubated with 0.8x Agencourt Ampure XP beads for 30 min, washed twice with 80% ethanol and resuspended in 6.4 μL nuclease free water. 9.6 μL of in vitro transcription mix is added (1.6 μL of each ribonucleotide, 1.6 μL T7 buffer, 1.6 μL T7 enzyme mix) and incubated at 37°C for 14 hours. After in vitro transcription, the aRNA  (amplified RNA) is size selected by incubating with 0.8x Agencourt RNAClean beads for 30 min, washed twice with 70% ethanol and resuspended in 16 μL nuclease-free water. Next, the aRNA is fragmented using fragmentation buffer (200mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc) at 94°C for 1.5 min and quenched by putting the reaction on ice and adding 2 μL 0.5 M EDTA. Fragmented aRNA is then purified using 1x Agencourt RNAClean beads as mentioned before and eluted in 16 uL nuclease-free water. After these steps, library preparation is performed as described in the scAba-seq protocol. scAba-seq [Mooijman et al. 2016 Nature Biotechnology]