title: GSM8720584: 4sU_ADE_d4G-_rep2; Mus musculus; OTHER
library_name: GSM8720584
library_construction_protocol: 4sU incubation was performed in ADE differentiation medium (ADEM; see PMID: 26840225) from the T75 flasks to be used for FACS sorting. To obtain the ADEM, 15 ml of ADEM was pipetted from each T75 flask, flasks were returned to the incubator and the extracted ADEM was pooled and centrifuged at 1000 g for 5 mins to remove cell debris. In the following steps, flasks were treated in the same order for every action taken in batches of up to 6 flasks to avoid timing differences between flasks. After aspiration, 5 ml pooled ADEM (negative control; 1-2 flasks per sort) or 5 ml pooled ADEM containing 500 μM 4sU were added and flasks were then returned to the incubator. After exactly 20 mins, flasks were aspirated, washed with 10 ml PBS (Merck, D8537), aspirated again and incubated for 3 min at 37°C in 2 ml per flask of attenuated trypsin (0.025% (v/v) trypsin (Thermo Fisher Scientific, 15090–046), 1.27 mM EDTA, and 1% (v/v) Chicken serum (Merck C5405) in PBS (Merck D8537)). Cells were then released from the flask by tapping and attenuated trypsin was inactivated by adding 8 ml ice-cold flow cytometry buffer (FC buffer - 10% FCS in PBS). Cell suspensions from all flasks with the same treatment in the batch were then pooled, pipetted repeatedly to obtain a single cell suspension, counted using a hemocytometer (Neubauer) and stored on ice. This process was repeated when required (for day 3 & day 4 samples) until a sufficient number of cells were obtained for the sorting procedure 100M cells). All centrifugation steps hereafter were performed at 400 g for 3 mins. Samples were centrifuged, aspirated, resuspended in ice-cold FC buffer at 1-1.5 x 107 cells/ml and passed through a cell strainer (Corning, 352235). Negative control cells were not sorted and kept on ice during the sorting procedure. 4sU-labelled cells were sorted on a BD FACSAriaIII into 5 ml Falcon tubes (Corning) at 4°C containing 1 ml of ice-cold FC buffer. After sorting 1 million cells, the tube was transferred to ice in the dark and replaced with a fresh 5 ml Falcon tube containing 1 ml of ice-cold FC buffer and this process was repeated until all 4sU-labelled cells were sorted. Samples were centrifuged, resuspended in 100 μl of ice-cold PBS and cells from the same population were pooled to obtain an equal number of cells for each population, equivalent to the population that generated the lowest number of cells and an identical number of cells was taken from the unsorted and unlabelled negative control sample. All samples were then transferred to 1.5 ml DNA LoBind tubes (Eppendorf, Enfield, CT 06082), added up to 1 ml using ice-cold PBS, centrifuged, aspirated and agitated to resuspend cells in the remaining PBS (<10 μl). Cells were lysed by adding 1 ml of Trizol reagent (Thermo Fisher Scientific) equilibrated to RT. After 5 mins of incubation at RT, samples were stored at -20 ºC. 4sU incorporated RNAs were isolated as previously described (PMID: 32439634; as adapted from PMID: 25811895). Ribosomal RNA was depleted from the purified RNA using the low-input RiboMinus eukaryote system v2 kit according to the manufacturer's instructions (Ambion A15027). Libraries were constructed using the NEBNext Ultra II directional RNA library preparation kit for Illumina according to the protocol for ribosome-depleted RNA and with an 11-min RNA fragmentation step (NEB E7760). Library PCRs were supplemented with 2× SYBR dye (Sigma S9430) so that amplification could be monitored by quantitative PCR on a Roche LightCycler 480. Individual libraries were pooled into two six-sample equimolar pools containing the indexes 3-8 (NEBNext multiplex oligos for Illumina, set 1, E7335) and deep-sequenced at BGI on a HiSeq4000 (paired-end 100-bp reads).